Page 1

Suppression of Methemoglobin Formation in Hemoglobin Vesicles

Using Peroxidase Activity of Methemoglobin

A Thesis

Presented to

Waseda University

March 2007

Tomoyasu ATOJI

Promoter:

Prof. Dr. Shinji Takeoka

Referees:

Prof. Dr. Hiroyuki Nishide

Prof. Dr. Kiyotaka Sakai

Preface

Since the discovery of the blood type classified into A, B, AB and O by

Landsteiner in 1900, the researches about blood were progressed. It was occupied the

important position in medicine. The requirement of blood supply on the wars was existed

in the background.

Transfusion are ranked as the transplantation of organ called blood, there are many

problems such as virus infection and compatibility. In addition to these problems, the

absolute shortage of the amount of donated blood is now serious problem. Now we are

facing the time of reconsideration of transfusion system in the viewpoint of medicine,

technology and administration.

It is considered that the inestimable benefits will be brought if blood substitutes are

developed and applied to clinical use. Some of modified hemoglobins (Hb) as blood

substitutes are now in clinical application. However, the safety and efficacies are

disputable. In particular, Hb molecule is essentially encapsulated by the erythrocyte

membrane for the control of physiological activities of Hb. Therefore, the safety modified

Hbs are not perfectly guaranteed even if the oxygen carrying ability is high in vivo.

The author’s group has developed Hb vesicles which encapsulate highly

concentrated and purified Hb solution within a phospholipid bilayer membrane as an

artificial oxygen carrier. The structure is similar to erythrocyte at the point of Hb

encapsulation though the size, shape and other parameters are difference.

In this thesis, I focused on the interaction of oxyHb in Hb vesicles with hydrogen

peroxide (H2O2), and the suppression of metHb formation in Hb vesicles. And from these

studies, I suggested the second generation Hb vesicles which had the H2O2 elimination

system by daring method.

The thesis would be benefit and contribute to the development of Hb vesicles.

Tomoyasu Atoji

i

Table of Contents

Preface

Table of Contents

Chapter 1:

Redox Activities of Heme Proteins and Outline of Artificial Oxygen Carriers

1

1. Introduction

2

2. Characteristics and reactivity of reactive oxygen species

2

2-1. Rule of ROS in living body

2

2-2. Electron states of ROS

2

2-3. Details of ROS

4

3. Redox activities of Hb and heme proteins

7

3-1. Characteristics of catalase

7

3-2. catalase activity of heme proteins

8

3-3. Redox activity of Hb

9

4. Development of artificial oxygen carriers

10

4-1. Cross-linked Hb

10

4-2. PEG-Hb

10

4-3. Poly-Hb

11

4-4. Albumin-heme

13

4-5. Perfluorocarbon emulsion

13

5. Hb vesicle as artificial oxygen carrier

14

References

16

Chapter 2:

Interaction of OxyHb or OxyHb Vesicles with H2O2

and Effect of Hb Encapsulation in Vesicles

23

1. Introduction

24

2. Purification of Hb solution and preparation of Hb vesicles

26

2-1. Purification of Hb solution from donated red blood cells

26

2-2. preparation of Hb vesicles

26

3. Interaction of oxyHb or oxyHb vesicles with H2O2

27

3-1. Experimental section

27

3-2. UV-vis measurement of interaction of Hb samples with H2O2

27

3-3. Changes of Hb components

29

ii

4. Lipid peroxidation of egg yolk lecithin by ferrylHb

33

4-1. Experimental section

33

4-2. Lipid peroxidation of EYL vesicles

34

5. Supremacy of Hb vesicles over acellular type HBOCs

35

References

37

Chapter 3:

Reduction of MetHb in Hb Vesicles

Using Membrane Permeability of Reductants

41

1. Introduction

42

2. Autoxidation mechanism of metHb formation from oxyHb

43

2-1. autoxidation of oxyHb43

2-2. Influence of pO2 to autoxidation

44

3. MetHb reduction system in red blood cells

45

3-1. Enzymatic reduction system

45

3-2. Non-enzymatic reduction system

46

4. Reduction of metHb by reductants under anaerobic condition

47

4-1. Experimental section

47

4-2. Structure of reductants

47

4-3. UV-vis measurement

48

5. Reduction of metHb by reductants under aerobic condition

49

5-1. Experimental section

49

5-2. UV-vis measurement

49

6. Autoxidation of reductants and inhibition of metHb reduction

50

6-1. Experimental section

50

6-2. Rate constants of autoxidation and metHb reduction of reductants 50

6-3. Inhibition of metHb reduction

51

7. Reduction of metHb in Hb vesicles

using membrane permeability of reductants

52

7-1. Experimental section

52

7-2. MetHb vesicles reduction under anaerobic condition

53

7-3. Rate constants of metHb reduction in Hb vesicles

54

7-4. MetHb vesicles reduction under aerobic condition

55

8. Effect of catalase co-encapsulation on metHb reduction in Hb vesicles 56

8-1. Experimental section

56

8-2. Effect of catalase co-encapsulation

57

9. Kinetics of metHb reduction in Hb vesicles

58

References

60

iii

Chapter 4:

Construction of H2O2 Elimination System

Using Peroxidase Activity of MetHb

63

1. Introduction

64

2. Reaction of metHb with H2O2 and generation of ferrylHb radical

66

2-1. Experimental section

66

2-2. Elimination of H2O2 by the metHb

67

2-3. UV-vis measurement

67

3. Preparation of ferrylHb radical and its radical mechanisms

69

3-1. Experimental section

69

3-2. UV-vis measurement

69

3-3. ESR measurement

70

3-4. Mechanisms of generation, isolation

and disappearance of ferrylHb radical

71

4. Temperature stability of ferrylHb radical

72

4-1. Experimental section

72

4-2. Conversion of ferrylHb radical

73

4-3. Autoreduction of ferrylHb radical

73

5. Reduction of ferrylHb radical by L-Tyr

74

5-1. Experimental section

74

5-2. Reduction of ferrylHb radical by L-Tyr (UV-vis measurement)

74

5-3. Reduction of ferrylHb radical by L-Tyr (ESR measurement)

75

5-4. Radical mechanism of ferrylHb radical

76

6. Analysis of L-Tyr in the reaction of metHb with H2O2

77

6-1. Experimental section

77

6-2. HPLC analysis of L-Tyr

77

6-3. Mechanism of diTyr generation

77

References

79

Chapter 5:

Application of H2O2 Elimination System by MetHb and L-Tyr

to the Hb Vesicles

83

1. Introduction

84

2. Stepwise injection of H2O2 to metHb/L-Tyr added oxyHb solution

85

2-1. Experimental section

85

2-2. Effect of metHb/L-Tyr system

86

3. Autoxidation of metHb/L-Tyr added oxyHb solution

87

3-1. Experimental section

87

iv

3-2. Effect of metHb/L-Tyr system

87

4. Preparation of the Hb vesicles containing metHb and L-Tyr

88

4-1. Experimental section

88

4-2. metHb/L-Tyr containing Hb vesicles ((metHb/L-Tyr) Hb vesicles)

89

5. Stepwise injection of H2O2 to a dispersion of (metHb/L-Tyr) Hb vesicles

90

5-1. Experimental section

90

5-2. Effect of metHb/L-Tyr coencapsulating to Hb vesicles

90

6. Autoxidation of (metHb/L-Tyr) Hb vesicles

91

6-1. Experimental section

91

6-2. Suppression of autoxidation

91

7. In vivo measurement of (metHb/L-Tyr) Hb vesicles

93

7-1. Experimental section

93

7-2. Effect in vivo

93

References

95

Chapter 6:

Extension of H2O2 Elimination System by MetHb

for the Various Applications

99

1. Introduction

100

3. FerrylHb radical reduction to metHb by various substrates

103

2-1. Experimental section

100

2-2. Conversion to metHb from ferrylHb radical

101

3. H2O2 elimination by the metHb/substrate system

103

3-1. Experimental section

103

3-2. Effect of metHb/substrate system

103

References

105

Chapter 7:

Conclusions and Future Prospects

107

1. Conclusions

108

2. Future prospects

109

Academic Achievements

110

Acknowledgement

113

Chapter 1

- 1 -

Chapter 1

Redox Activities of Heme Proteins

and Outline of Artificial Oxygen Carriers

Contents

1. Introduction

2. Characteristics and reactivity of reactive oxygen species

2-1. Rule of ROS in living body

2-2. Electron states of ROS

2-3. Details of ROS

3. Redox activities of Hb and heme proteins

3-1. Characteristics of catalase

3-2. catalase activity of heme proteins

3-3. Redox activity of Hb

4. Development of artificial oxygen carriers

4-1. Cross-linked Hb

4-2. PEG-Hb

4-3. Poly-Hb

4-4. Albumin-heme

4-5. Perfluorocarbon emulsion

5. Hb vesicle as artificial oxygen carrier

References

Chapter 1

- 2 -

1. Introduction

In living body, various redox reactions of proteins are occurring for the various

purposes. In particular, heme proteins such as catalase, cytochrome P450, and peroxidases

play important rules for the redox reactions. In this chapter, the redox reactions of these

proteins were summarized. However, “wrong” redox reactions are also occurring in our

living body. It is due to reactive oxygen species (ROS). ROS such as hydrogen peroxide

(H2O2), superoxide anion radical (O2

-*), hydroxyl radical (OH-*), and nitric monoxide

(NO) are considered as aging. Then, we focused on the redox activity of heme proteins

with ROS in this chapter. Characteristics of ROS were also summarized in this chapter.

Our purpose of this thesis is the prolongation of oxygen carrying ability of

hemoglobin (Hb) vesicles, which encapsulate highly concentrated and purified Hb solution

(Hb purification; > 99.9 %, concentration; 36 g/dl) within a phospholipid bilayer

membrane as an artificial oxygen carrier 1-5. Experimental contents were described after

this chapter, here we summarized the developments of various types of artificial oxygen

carriers. To know the various types of artificial oxygen carries will lead to the

development and advance of our material.

2. Characteristics and reactivity of reactive oxygen species

2-1. Rule of ROS in living body

It is considered that ROS in the living body play the important rule for aging,

diseases and death. Like this, ROS administrate the lifespan of all livings exposing O2.

And we cannot avoid the fortune, and we are necessitated to accept it.

2-2. Electron states of ROS

O2 molecule mainly forms the following three state; triplet O2 (3O2), singlet O2

(1O2), and superoxide anion radical (O2

-*). Furthermore, 1O2 is classified into two states by

the energy state 6. 3O2 is normal O2 including in air. It is the most stable state of O2 in the

viewpoint of thermodynamics. O2

-* is well known as one of the ROS. The electron states

are shown in Figure 1-1 and 1-2. Only the electron states of πx* orbital are difference each

other. Two kinds of singlet O2 are defined as ROS, they are immediately quenched to

triplet O2 after generation in air. However, once in a great while, the singlet oxygen reacts

Chapter 1

- 3 -

with other ROS such as NO and OH-* to form more strong ROS (e. g. peroxynitrite

(ONOO-), hydroperoxide (HOO-*)).

Figure 1-1 Electron spin states and the πx* states of various O2 state. Only the electron

states of πx* orbital are difference each other.

Figure 1-2 The πx* orbitals of various O2 state.

Chapter 1

- 4 -

2-3. Details of ROS

The various ROS and their derivatives, and their molecular formulas are shown in

Table 1-1.

superoxide anion radical (O2

-*)

Superoxide anion radical is generally shown as “O2

-*”. It is the one electron

reduction state of O2 molecule, to say in other words, it is activated state that O2 molecule

obtains one electron. Therefore, it is defined as ROS. O2

-* is the first ROS product

generated from O2 molecule, it plays a rule as the precursor of various ROS in addition to

the reactivity itself. It also reacts with nitric monoxide (NO) 7.

O2

-* is generated by the various mechanisms or reactions involving O2 in living

body. In the NADPH-depending cytochrome P450 system in cell membranes, O2

-*

continuously generates under physiological condition. Furthermore, the ADPH-depending

cytochrome P450 reductase reduces paraquat (methylviologen) or quinine type anticancer

drugs such as adriamycin in the presence of O2, O2

-* is generated as the results of the

redox cycles. And O2

-* is also generated by the one electron transfer to O2 molecules by

the semi-quinone form of ubiquinone generated by the one electron reduction of

ubiquinone in the electron transfer system of mitochondria 8.

O2

-* is stable in the nonprotic solvents. On the other hand, it is dismutated to

hydrogen peroxide (H2O2) by protonation in aqueous solution. Furthermore, the one

electron loss from O2

-* to form O2 also occurred in aqueous solution.

Table 1-1 Various ROS and their derivatives,

and their molecular formulas.

Chapter 1

- 5 -

hydrogen peroxide (H2O2)

H2O2 is one of ROS, however, it does not have unpaired electron. Therefore, the

reactivity of H2O2 itself are not so much high compared with radical species such as O2

-*

and OH-*. The important point is the generation of OH-* by Fenton reaction described in

next part. Almost H2O2 generated in vivo is formed from the dismutation of O2

-*. It is

comparatively stable under physiological conditions. It is easily decomposed by the

reaction with metal or the irradiation of light, X-ray and gamma-ray to form OH-* 9.

H2O2 smoothly permeates cell membrane composing from lipid bilayer membrane.

Namely, the cell membrane is not the obstruction against the H2O2 attack to the cell inside.

It is the same on the membrane composing Hb vesicle 10, 11. On the other hand, O2

-* is not

able to permeate lipid bilayer membrane 11.

H2O2 is generated by the two electron reduction of O2 molecules, one electron

reduction of O2

-*, or the oxidation reactions with various compounds (e. g. alcohols,

sugars, etc).

The details of reactivity of H2O2, in particular with Hb, were described in Chapter

2-7, and they were main contents of this thesis.

hydroxyl radical (OH-*)

hydroxyl radical is generally shown as “OH-*”. It belongs to the ROS group which

has very high radical reactivity. Therefore, it is considered that the majority of the damage

of living body by ROS in vivo is due to OH-*. OH-* is strong oxidant because it forcibly

intercepts one electron from various compounds. Furthermore, it causes the hydrogen

withdrawing reaction and the addition reaction to the double bonding part and phenyl ring

of various compounds 12. Their reaction rate constants are generally high. However, the

lifetime of OH-* is too short to reach to the distant positions from generation position of

OH-*. OH-* indiscriminately reacts with sugars, lipids, proteins and DNA, the reactions do

not has specificities.

OH-* is generated in the reaction of H2O2 with metal iron or X-ray irradiation to

H2O molecule in vivo. In particular, the OH-* generation by the reaction of H2O2 with

ferrous iron is well known as Fenton reaction. It is shown as follows.

Fe2+ + H2O2

Fe3+ + OH-*+ OH-

（k = 76 M-1s-1）

Chapter 1

- 6 -

OH-* is generated from H2O molecule in cell by the irradiation of X-ray or

gamma-ray to tumors for the therapy. The almost DNA damages in cells or cell membrane

damages are considered to be caused by OH-*. The damages are deeply related to aging,

diseases and death 13.

Singlet oxygen (1O2)

Singlet oxygen is generally shown as “1O2”. 1O2 is one of ROS, however, it does

not have unpaired electron like H2O2. Therefore, 1O2 itself is not free radical. 1O2 has the

electron structure that two electron spins at πx* and πz* of triplet oxygen (3O2) are in

anti-parallel each other. It is shown in Figure 1-1. Furthermore, it is classified into two

types; two electrons in same orbital (1O2(1Δg)), and two electrons in difference orbitals

(1O2(1Σg)). The lifetime of the former is extremely short, it immediately converts to the

latter. Therefore, 1O2 generally indicates the latter 14.

The energy of 1O2 is 22.5 kcal/mol larger than that of 3O2. So, 1O2 is

thermodynamically activated state compared with 3O2. 1O2 reacts with unsaturated fatty

acid to form peroxide in the lipid molecule, and it also reacts with hydroxyltryptophan to

form O2

-*.

1O2 is generated by the decomposition of peroxides, photosensitization reactions,

or ultrashort wave discharge. In the living body, 1O2 is also generated by the reaction of

H2O2 with hypochlorite.

H2O2

+ ClO-

H2O + Cl- + 1O2

Moreover, the reaction of two lipid peroxy radical molecules generates 1O2

15.

nitric monoxide

Nitric monoxide is generally shown as “NO”. It is also shown as “NO*” because

nitric oxide has unpaired electron. It is known as toxicity molecule including in the

cigarette smoke and manufacturing smoke from factories. However, it was proven that NO

was a vasodepressor substrate from endothelial cells 16, it was cleared that NO played

various rules under physiological condition in vivo 17.

NO is synthesized from the arginine by NO synthase in neurocytes or endothelial

cells in vivo. NO easily reacts with O2

-* to form peroxynitrite (ONOO-). The toxicity of

peroxynitrite is stronger than NO, it is the causes of the damages of proteins, lipids, DNA,

or cells themselves.

Chapter 1

- 7 -

NO + O2

-*

ONOO-

The NO production, reaction, and elimination systems are very important for living body,

and the mechanisms are very interesting.

3. Redox activities of Hb and heme proteins

3-1. Characteristics of catalase 18-20

Catalase widely distributes over natural, and it is tetramer composed from four

units. The molecular weight is 220 kDa – 260 kDa, it is due to the source. The center of

the reactivity is protoheme as well as Hb, it includes one protoheme per unit. Catalase

catalyzes the following dismutation reaction (a).

2H2O2

H2O + O2

(a)

From the reaction (a), it is understood that catalase eliminates two H2O2 molecules in one

cycle. It is shown as follows;

catalase(Fe3+) + H2O2

Compound I + H2O

(b)

Compound I + H2O2

catalase(Fe3+) ＋ O2

＋ H2O (c)

When the Compound I reacts other substrates, the reaction is,

Compound I + AH2

catalase(Fe3+) ＋ O2

＋ H2O (d)

Various theories about structure of Compound I was suggested, in present, it was

agreed that the transition state including π-cation radical of porphyrin possessing ferryl

state iron (Fe4+) was the compound I of catalase by the physico-chemical measurements by

Dolphin et al. in 1971. Therefore, reaction (b) and (c) are expressed as follows.

Por(Fe3+) + H2O2

Por(Fe4+=O*) + H2O

(e)

Por(Fe4+=O*) + H2O2

Por(Fe3+) + O2

+ H2O (f)

In generally, the facility of heme proteins is the redox activities between the

oxidized and reduced states. Catalase forms the four redox states. These states are shown

in figure 1-3. Catalase usually stays in the ground state, which heme iron is ferric state.

High oxidative states than ferric state defined as Compound I, and II are the ferryl radical

and ferryl state of catalase, respectively. Compound III is very specific state of catalase,

which is perferryl state (Fe5+). However, it is apparent state in theory, practically the

Chapter 1

- 8 -

electron is delocalized in porphyrin ring or peptide chain of catalase 21. The reaction rate

constant of reaction (e) is 1.7 x 107 M-1s-1, it is quite large.

3-2. Catalase activity of heme protein

Catalase activity is calculated from the absorption change at 240 nm of UV region.

The activity is defined as “1 unit of catalase is the ability that 1 mol of H2O2 is eliminated

by it for 1 minute at 25 oC” 22.

The definition is able to apply to other heme proteins, it is the catalase activity of

heme proteins. It is also called pseudo-catalase activity or catalase-like activity of heme

proteins.

The inhibition factors of catalase activity are classified into two patterns. One is

when the ligand molecule is CN- or F-. They inhibit the reaction by the occupation of H2O2

binding site. The other is catalase inactivation by the reaction of high oxidized catalase

with inhibitor. For example, the particular kinds of phenol compounds, alcohols and

quinines inhibit the catalase activity by the one electron oxidation of Compound I to

Compound II 23.

k1

1.7×107 M-1s-1

k5

1.1×10-2 s-1

k2

1.0×107 M-1s-1

k6

2.7×10-4 s-1

k3

5.0×106 M-1s-1

k7

6.1×104 M-1s-1

k4

2.0×105 M-1s-1

k9

2.0×102 M-1s-1

rate constant k (pH7.0, 25 oC)

k1

1.7×107 M-1s-1

k5

1.1×10-2 s-1

k2

1.0×107 M-1s-1

k6

2.7×10-4 s-1

k3

5.0×106 M-1s-1

k7

6.1×104 M-1s-1

k4

2.0×105 M-1s-1

k9

2.0×102 M-1s-1

rate constant k (pH7.0, 25 oC)

Figure 1-3 Electron states of various forms of catalase and reaction rate constants.

Chapter 1

- 9 -

3-3. Redox activity of Hb

The ferryl radical state (Compound I) of Hb, which is mainly induced by the

reaction with H2O2, is known to be strong oxidants for several biomolecules such as

tocopherol (and its analog Trolox C) 24, 25 and ascorbic acid 26, 27, and are reduced to the

respective ferric states (metHb and metMb) by the acceptance of two electrons from these

biomolecules. Oxidative products of substrates and their related products were detected in

human erythrocytes and were thought to be produced by the proteolysis of the ferrylHb

radical generated by H2O2

28. Thus, the ferryl radical of Hb is unstable due to their high

reactivity. Thus, the ferryl radical of Hb is unstable due to their high reactivity. The

enzymatic pseudo-catalase activity is generally shown as follows;

Hb(Fe3+) + H2O2

Hb(Fe4+=O*) + H2O (g)

Hb(Fe4+=O*) + H2O2

Hb(He3+) + H2O + O2

(h-1)

Hb(Fe4+=O*)

denaturation, heme release

(h-2)

Because ferrylHb radical is very unstable, therefore, equation (h-2) is mainly

occurred in the reaction. This is the reason of that metHb is not worked as H2O2

elimination enzyme like catalase. Thus, the ferryl radical of Hb is unstable due to their

high radical reactivity. Nagababu et al. reported a reaction intermediate generated from the

oxoferryl state of Hb (Hb(Fe4+=O)) in the process of heme degradation and the proteolysis

of Hb by H2O2, and named it “rhombic heme” 29.

Chapter 1

- 10 -

4. Development of artificial oxygen carriers

4-1. Cross-linked Hb

Hb molecule is composed from the four sub units (α1, α2, β1, β2), if it is directly

administrated without modification or encapsulation, Hb molecule is dissociated to

αβ-units dimer in vivo, it causes grave nephrotoxicity by the filtration from kidney 30.

Cross-linked Hb (XLHb) has the structure that intramolecular two α units of Hb (α1,2

Lys-99) are cross-linked by bis(3,5-dibromosalicyl) fumarate (DBBF) as a cross linker for

the prevention from the dissociation 31. The structural image is shown in Figure 1-4.

XLHb has the characteristic of long circulation time in bloodstream following the

modification for prevention of dissociation. It was developed by Baxter Healthcare Corp.

(USA) and U.S. army. In 1991, Baxter Healthcare Corp. acquired the license of clinical

trial of the XLHb from US FDA, the trials about safety and efficacies to trauma were

proceeded to the phase III. However, the reports about the side effects such as high blood

pressure by vasoconstriction and deformity of gut increased, the clinical trial was stopped

in September, 1998. Now, they have been developing the recombinant Hb which has a

control facility of NO binding as a second generation32 (now in pre-clinical trial).

Abdu I. Alayash at US FDA evaluates the safety and the cytotoxicity of modified

Hb (in particular XLHb) in the reaction with ROS such as H2O2, NO, peroxinitrite

(ONOO-) from the standpoint of biochemistry 30, 31, 33. Furthermore, he continues to

suggest the evaluation standards and items about efficacies and safety of artificial oxygen

carrier from the standpoint of administration (US FDA).

4-2. PEG-Hb

PEG-PLP-Hb is formed form the Schiff base in N-terminal of b unit of Hb

molecule with pyridoxal 5’-phospate (PLP) for the allosteric effector. The surface of the

molecule is modified by the PEG for the prolongation of circulation time ion bloodstream.

The structural image is shown in Figure 1-5.

α

α

β

β

α

α

β

β

Figure 1-4 Structural image of XLHb. Two Lys-99

residues of α1 and α2 units are cross-linked by DBBF

as a cross-linker.

Chapter 1

- 11 -

PEG-PLP-Hb was first developed by Ajinomoto Co. Ltd., at present, Curacyte

Health Sciences (Germany) continues to develop the PEG-PLP-Hb as Pyridoxalated Hb

Polyoxyethulene (PHP) 34. It is now in the phase III clinical trial for the clinical application

to the oxygenation of tumor and ichorrhemia treatment. However, it is considered that the

application to the red blood cell substitute is difficult. Enzon Inc. (USA) developed the

PEG-Hb using Hb from bovine, but they were necessitated to stop the development by the

patent problem.

In present, PEG-Hb is developed by R. M. Winslow et al. at Sangart inc. (USA)

and M. Intagletta at University of California San Diego. Their group solved the problems

on Enzon Inc. (patent problems, etc) by the method of “PEGylation”, which is the

technique of surface modification of Hb molecule by maleimide-PEG. The PEG reacts

with six SH groups come from cysteine residues of Hb surface. The PEG-Hb called

Hemospan has high viscosity (3.5 cp at 2 g/dl), high colloid osmotic pressure (20 mmHg),

and high oxygen affinity (P50: 10 – 12 mmHg) 35. The high P50 enables to supply oxygen

to the periphery tissues exposed to hypoxemia. From these characteristics of EG-Hb, they

suggest the high efficacy of tissue oxygenation exposed to hypoxemia by low amount

administration. Furthermore, the report by B. Kjellstrom (Kalorinska Inst., Sweden) was

proven that the safety and no side effects were confirmed by the administration of

Hemospan from the observation of microcirculation and measurement of biochemical

profiles using hemorrhagic shock model of porcine 36. In present, the phase I clinical trial

of Hemospan has been proceeding in Sweden.

4-3. Poly-Hb

Poly-Hb has a polymer structure that Hb molecules are cross-linked between

intra- and intermolecules. By increase of molecular weight and size, the decrease of the

grave nephrotoxicity by the filtration from kidney and the deformity of gut are achieved

37-40. The structural image of poly-Hb is shown in Figure 1-6.

Figure 1-5 Structural image of PEG-PLP-Hb.

The surface is modified by PEG

Chapter 1

- 12 -

Biopure Corp. (USA) has performed the most advanced research about poly-Hb.

They acquired the license of clinical trial of the poly-Hb cross-linking bovine Hb by

glutaraldehyde called “Hemopure (HBOC-201)” from Drug Administration of South

Africa, where it is indicated for use in adult surgical patients who are acutely anaemic, and

is used to eliminate, delay or reduce the need for allogeneic red blood cells, in 31th July,

2002. HBOC-201 has characteristics of the long-term preservation for three years at 30 oC,

the high performance of oxygen binding-dissociation ability equaling to erythrocyte, and

the 19 hr of half life in blood stream in human 41-44. In the report in 2003, the grave side

effects were not confirmed from the administration tests to over 800 patients over 20 kinds

of clinical trials 45. Multi-centre, randomised, Phase III, controlled trials have

demonstrated the safety and efficacy of HBOC-201. A Biologics License Application for

HBOC-201 is currently under review by US FDA.

Northfield Laboratories Inc. (USA) also has developed the poly-Hb called

“polyheme”. Polyheme is a human hemoglobin-based temporary oxygen-carrying red

blood cell substitute in development for the treatment of life-threatening blood loss when

an oxygen-carrying fluid is required and red blood cells are not available. Polyheme has

the characteristics of the simultaneously restores lost blood volume and hemoglobin levels,

universally compatible (does not require typing or cross-matching before infusion),

immediately available, extending shelf life in excess of 12 months, and manufacturing

from human red blood cells using steps to reduce the risk of viral transmission 46-50.

Polyheme has a 50 g of Hb per unit, it enables to carry the same amount of oxygen

compared with red blood cells. In November 2001, the permission of the clinical trial

license was rejected by US FDA, however in March 5th 2003, they acquired the license of

phase III clinical trial of Polyheme.

Hemosol Inc. (Canada) has developed the human raffinose poly-Hb cross-lined

between the Hb molecules called “Hemolink”, the safety and efficacies were already

Figure 1-6 Structural image of poly-Hb. Hb

molecules are cross-linked between intra- and

intermolecules.

Chapter 1

- 13 -

confirmed from the administration to over 600 patients in phase I and II clinical trial. Now,

Hemolink is under phase III clinical trial 51-53.

4-4. Albumin-heme

Albumin-heme has the structure that tertaphenylporphyrin derivatives or

protoporphyrin derivatives concluding ferrous iron are involved in recombinant human

serum albumin (rHSA). It has been developed by Tsuchida’s group at Waseda University

(Japan), it is defined as the total synthesized artificial oxygen carrier. The hydrophibic

position of albumin molecule enables to form the stable oxygen complex of heme, and the

oxygen affinity and oxygen half life are able to control by the molecular structure of

involved heme 54-57. Now the evaluations of efficacies and safety toward clinical trial have

been proceeded.

4-5. Perfluorocarbon emulsion

Perfluorocarbon (PFC) as an artificial oxygen carrier is the emulsion composed

from the fluorocarbon compounds which has no physiologic activities. It utilizes the

physical oxygen solubility to PFC emulsion. The kinds of PFC emulsions have very high

oxygen solubility, it achieves to 40 – 50 vol% to the emulsion volume. The picture of PFC

(Fluosol-DA20) is shown in Figure 1-7.

In the development of PFC emulsion, Green Cross Corp. (Japan) acquired the

license of sales of the PFC emulsion called “Fluosol-DA20” from US FDA. However the

use was limited to the treatment after percutaneous transluminal angioplasty. Furthermore,

the oxygen carrying ability was one of fifth compared with human blood in addition to the

low ability for preservation. Finally from these reasons, the manufacturing and sales were

foreclosed in 1993.

Figure 1-7 Picture of PFC (Fluosol-DA20).

Chapter 1

- 14 -

Riess J. G. and Keipert K. et al. at Alliance Pharmaceuticals Inc. (USA) developed

the second generation type of PFC emulsion. It was given the thermostability by the PEG

modification of emulsion surface 58. The PFC was acquired the license for clinical

application to supersonic diagnosis by PFC microbubble. Furthermore, it was expected to

use the dilute solution for self-blood therapy. However, the development was necessitated

to interrupt by the financial problem.

Maevsky E. I. at Inst. Theoretical and Experimental Biophysics (Russia) has

developed the PFC emulsion called Perftoran using proxanol 268 as a stabilizer 59. It has

the characteristics of good oxygen carrying ability to microcirculation system due to the

diameter of 70 nm. It was administrated to over 200 patients in various clinical trials

(trauma, hemorrhage shock, fat embolism, cerebral edema, etc.), the efficacy to early step

of hemorrhage and the oxygenation of ischemia were confirmed until the end of 2000.

Kraft M. P. and Riess J. G. et al. reported the new PFC emulsion which had the

high stability and little particle diameter (80 nm) 60. The characteristics were come from

the application of carbon hydride including fluorine to perfluorooctylbromide emulsion.

The stability of preservation was 6 months at 25 oC, it was dramatically long compared

with Fluosol-DA20.

Matsumoto S. at Kyoto University (Japan) showed the efficacies of the bistratal

phase system of PFC/UW (Univ. of Wisconsin Solution) for the preservation of the organs

for transplantation (pancreas and nesidioblast) 61. In USA, it is under clinical use.

5. Hb vesicles as artificial oxygen carrier

Hb vesicles have been studied by our group at Waseda University. We have

developed Hb vesicles which encapsulate highly concentrated and purified Hb solution

(Hb purification; > 99.9 %, concentration; 36 g/dl) within a phospholipid bilayer

membrane as an artificial oxygen carrier 1-5, 10, 11. The picture and structural image are

shown in Figure 1-8.

The particle diameter is controlled to 250 + 30 nm, the particle surface is modified

by PEG-DSPE. It is enabled by the application techniques of the molecular assembly

strictly controlled. P50 is controlled by the co-encapsulating PLP as an allosteric effector 1.

Hb vesicles have the characteristic of the long-term preservation at room temperature (two

Chapter 1

- 15 -

years preservation), the colloidal osmotic pressure and viscosity are able to be controlled

to the physiological condition 2-3.

Now, the studies toward the clinical trial have been performed by the group

organized by Waseda University, Keio University, Oxygenix Co. Ltd., Nipro Corp, and

related field’s researchers and companies. The details of Hb vesicles were described in

chapter 2-7.

Diameter: 250 + 30 nm

Hb vesicle

Hb vesicle dispersion

PEG modification

>36 g/dl Hb solution

(Hb >99.9 %)

Phospholipid

bilayer menbrane

Diameter: 250 + 30 nm

Hb vesicle

Hb vesicle dispersion

PEG modification

>36 g/dl Hb solution

(Hb >99.9 %)

Phospholipid

bilayer menbrane

Figure 1-8 Picture of Hb vesicle dispersion and Hb vesicle.

Chapter 1

- 16 -

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Chapter 1

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29. Nagababu, E., Ramasamy, S., Rifkind, J. M., Jia, Y., and Alaysh, A. I., Site-specific

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30. D'Agnillo、F., and Alayash, A. I., Redox cycling of diaspirin cross-linked hemoglobin

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36. Heinius, G., Wladis, A., Hahn, R. G., and Kjellstrom, B. T., Induced hypothermia and

rewarming after hemorrhagic shock, J. Surg. Res. 108 (2002) 7-13.

37. Caswell, J. E., Strange, M.B., Rimmer, D. M. 3rd, Gibson, M. F., Cole, P., and Lefer,

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Resuscitation with a blood substitute causes vasoconstriction without nitric oxide

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39. Sampson, J. B., Davis, M. R., Mueller, D. L., Kashyap, V. S., Jenkins, D. H., and

Kerby, J. D., A comparison of the hemoglobin-based oxygen carrier HBOC-201 to

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bovine blood with varying lead concentrations, Anesth. Analg. 96 (2003) 1813-20,

41. Standl, T., Freitag, M., Burmeister, M. A., Horn, E. P., Wilhelm, S., and Esch, J. S.,

Hemoglobin-based oxygen carrier HBOC-201 provides higher and faster increase in

oxygen tension in skeletal muscle of anemic dogs than do stored red blood cells, J.

Chapter 1

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Vasc. Surg. 37 (2003) 859-65.

42. Ortegon, D. P., Davis, M. R., Dixon, P. S., Smith, D. L., Josephs, J. D., Mueller, D.

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without brain injury, J. Trauma 61 (2006) 1085-99.

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45. Levy, J. H., The use of haemoglobin glutamer-250 (HBOC-201) as an oxygen bridge

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48. Gasthuys, M., Alves, S., and Tabet, J. C., N-terminal adducts of bovine hemoglobin

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3372-3378.

49. Johnson, J. L., Moore, E. E., Gonzalez, R. J., Fedel, N., Partrick, D. A., and Silliman,

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57. Nakagawa, A., Komatsu, T., Iizuka, M., and Tsuchida E. Human serum albumin

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Chapter 1

- 22 -

Chapter 2

- 23 -

Chapter 2

Interaction of OxyHb or OxyHb Vesicles with H2O2

and Effect of Hb Encapsulation in Vesicles

Contents

1. Introduction

2. Purification of Hb solution and preparation of Hb vesicles

2-1. Purification of Hb solution from donated red blood cells

2-2. preparation of Hb vesicles

3. Interaction of oxyHb or oxyHb vesicles with H2O2

3-1. Experimental section

3-2. UV-vis measurement of interaction of Hb samples with H2O2

3-3. Changes of Hb components

4. Lipid peroxidation of egg yolk lecithin by ferrylHb

4-1. Experimental section

4-2. Lipid peroxidation of EYL vesicles

5. Supremacy of Hb vesicles over acellular type HBOCs

References

Chapter 2

- 24 -

1. Introduction

Enormous efforts have been made to develop red blood cell substitutes for clinical

applications in recent years 1-3. Potential benefits of red blood cell substitutes are their

infusion in emergency situations without concern for blood type, virus infections, and

long-term storage. Hb-based oxygen carriers (HBOCs), which have been developed so far,

are generally classified into two types: one is the acellular-type modified Hb molecules

such as intramolecularly cross-linked Hb 4, recombinant cross-linked Hb 5, polymerized

Hb 6, and intramolecularly polymer-conjugated Hb 7. Some of these Hb modifications are

currently in the final stage of clinical trials. However, clinical trials have revealed some

side effects such as hypertension relating to vasoconstriction, which have been extensively

reported in vivo and in vitro 8, 9. Another is the cellular-type Hb such as Hb vesicles which

are being developed by our group 10 or liposome-encapsulating Hb 11. They have a

vesicular structure in which concentrated Hb molecules are encapsulated, like red blood

cells. Though the Hb vesicles have not yet been clinically studied, their safety and efficacy

have been confirmed in comparison with Hb modifications in order to insist on the

benefits of the cellular structure 12-17. From safety tests involving microvascular responses

12, 13 and heme detoxification in liver 17, we have clarified the sufficient oxygen

transporting ability comparable with red blood cells in 40 % hemorrhage shock 14 and

90 % exchange transfusion 16.

Recently, after the administration of the Hb modifications, it has been noted that

heme-mediated reactions such as ligand coordinations and redox reactions could cause

organ dysfunction and/or tissue damage 18, 19. Especially, redox reactions may affect the

physiological protection against reactive oxygen species 20. The oxidation of oxyHb by

H2O2 is known to generate ferrylHb and metHb accompanied by heme degradation and the

release of free iron 21. Furthermore, during the autoxidation of oxyHb to metHb, reactive

oxygen species such as superoxide, hydrogen peroxide, and the hydroxyl radical are

generated to damage not only the remaining oxyHb but also living cells and organs 22-24.

Especially, ferrylHb is known to be a potent oxidant which catalyzes the peroxidation of

lipids comprising the biomembrane and other biomaterials 25, 26. In normal human plasma,

the concentration of H2O2 is 4 - 5 μM 27 and elevates to 100 - 600 μM under inflammatory

28 or ischemia/reperfusion conditions 29, in fact, ferrylHb can be found both in the red

blood cells 30 and in the endothelial cells model after hypoxia reoxygenation 31, 32. Several

in vitro studies suggest that free radicals or degradation products catalyzed by ferrylHb

Chapter 2

- 25 -

could damage the endothelial cells in the presence of acellular-type Hb modifications.

Hb-mediated cytotoxicity via ferrylHb is one of the important safety issues of HBOCs 20,

33-36. On the other hand, in the cellular-type Hb vesicles 37, reactive oxygen species

generated within the Hb vesicles during metHb formation were completely consumed by

Hb (unpublished data). Though such a reaction leads to Hb oxidation, no reactive oxygen

species have been detected outside the vesicles.

It was proven that the main cause of metHb formation of Hb vesicles was H2O2

using the study of catalase-coencapsulating Hb vesicles 38. The result is shown in Figure

2-1.

In this chapter, we describe the chemical reactions between H2O2 and Hb (acellular

type), or H2O2 and Hb vesicle (cellular type) and demonstrate the influence of the resulting

ferrylHb on the peroxidation of unsaturated lipids, which were added to the system as egg

yolk lecithin vesicles to clarify the advantage of Hb vesicle over acellular-type Hb

concerning heme-mediated cytotoxicity via ferrylHb. Furthermore, we also mentioned to

the mechanism of metHb formation of oxyHb or oxyHb vesicles in the reaction with

H2O2.

0

20

40

60

80

100

0

5 10 15 20 25 30 35 40

Time (hr)

metHb

(%

)

50

HbV 1

HbV 6

HbV 4

HbV 3

HbV 2

HbV 5

HbV 7

0

20

40

60

80

100

0

5 10 15 20 25 30 35 40

Time (hr)

metHb

(%

)

50

HbV 1

HbV 6

HbV 4

HbV 3

HbV 2

HbV 5

HbV 7

Figure 2-1 Changes of metHb percentages in the catalase-coencapsulating Hb

vesicles in vivo. The concentrations of coencapsulating catalase were 1, 0

unit/ml (control), 2, 2.8x103 unit/ml, 3, 8.4x103 unit/ml, 4, 1.7x104 unit/ml, 5,

2.8x104 unit/ml, 6, 4.2x104 unit/ml, and 7, 5.6x104 unit/ml.

Chapter 2

- 26 -

2. Purification of Hb solution and preparation of Hb vesicles

2-1. Purification of Hb solution from donated red blood cells

Hb was purified from outdated human red blood cells provided by Japanese Red

Cross. The red blood cells were washed three times with saline by centrifugation (2000g,

10 min) and concentrated by the removal of the supernatant. They were hemolyzed by the

addition of the equal volume of water for injection, and then the stroma were removed by

ultrafiltration (cutoff Mw 1000 kDa, Biomax-1000V, Millipore Co., Ltd., Bedford). The

ligand exchange from O2 to CO was carried out for the stroma-free Hb solution by CO gas

flowing over the stirred solution. The proteins other than HbCO were denatured by heat

treatment at 60 oC for 12 hr and removed as precipitates. The HbCO solution was

fractionated using ultrafiltration filters with a cutoff molecular weight between 1000 kDa

and 8 kDa (Biomax-8V, Millipore), followed by concentration with an 8 kDa ultrafilter.

The all purified Hb in this thesis was prepared by this method.

2-2. preparation of Hb vesicles

Pyridoxal 5′-phosphate (PLP, Merck, Whitehouse Station) as an allosteric effector

was added to the HbCO solution (40 g/dl) at a 3/1 molar ratio of PLP to Hb. Presome

PPG-I [1,2-dipalmitoyl-sn-glycero-3-phosphatidylchorine (DPPC) / cholesterol /

1,2-dipalmitoyl- sn-glycero-3-phosphatidylglycerol (DPPG), (Nippon Fine Chemical,

Osaka)] powder was mixed with the HbCO solution, and the mixture was stirred at 4 °C

for 12 hr. The resulting dispersion of multilamellar vesicles were extruded through

membranefilters (Fuji Film Co., Tokyo) with a Remolino (Millipore). The Hb vesicles

with an average diameter of 250 + 20nm were obtained after extrusion through the

membrane filter with 0.22 μm pore size. After the separation of unencapsulated Hb by

ultracentrifugation (10000g, 60 min), the precipitate of the Hb vesicles was redispersed

into saline.

1,2-Distearoyl-sn-glycero-3-phosphatidylethanolamine-N-PEG (PEG

molecular weight was 5000, Sunbright DSPE-50H, H-form, NOF Co., Tokyo) was added

to the dispersion of the Hb vesicles until its concentration to the membrane components

became 0.3 mol %. The mixture was incubated at 37 °C for 2 hr for the surface

modification with PEG-DSPE. Finally, unincorporated DSPE-50H was removed by

ultracentrifugation (10000g, 60 min) as a supernatant.

Chapter 2

- 27 -

Note: Now, the lipid mixture “Presome PPG-I” is not used for the preparation of Hb

vesicles. At present, mixture lipid for Hb vesicles was composed from DPPC / cholesterol

/ dipalmitoylethanolamine (DPEA) added 0.3 mol% of PEG-DSPE. All experiments of

this chapter was performed in the time that Presome PPG-I was used for the preparation of

Hb vesicle.

3. Interaction of oxyHb or oxyHb vesicles with H2O2

3-1. Experimental section

After the decarbonylation of HbCO (HbCO to HbO2) by the irradiation of visible

light to theliquid film, the oxyHb solution and the oxyHb vesicle dispersion containing

deferoxamine mesylate (DFO, 1.6 mM) were used as Hb samples. Reaction of the Hb

samples ([heme] =20 μM) with H2O2 at various ratios in phosphate buffer (pH7.4, at

37 °C) was assessed by repetitive scanning of a visible region from 450 to 700 nm at 2

min intervals by using an UV-vis spectrometry (V-570, Jasco, Tokyo). The concentration

of H2O2

was determined spectrofluorometrically by measuring the amount of

6,6’-dihydroxy-[1,1’-biophenyl]-3,3’-diacetic acid (DBDA, Ex: 317 nm, Em: 405 nm),

generated by the horseradish peroxidase (HRP)-catalyzed reaction of

p-hydroxyphenylacetic acid (HPA) with H2O2. The final concentrations of HRP and HPA

were 4 μM and 6 mM, respectively, and the DBDA concentration was calculated after

separating the Hb samples by centrifugal filtration (cutoff 5 kDa , Ultrafree-MC, Millipore,

Bedford). It is called HRP method 39.

3-2. UV-vis measurement of interaction of Hb samples with H2O2

Figure 2-2 shows the spectral change in the Hb solution and Figure 2-3 shows the

spectral change in the Hb vesicle dispersion, both of which have a heme concentration of

20 μM, after the addition of H2O2 of three different concentrations: (a) 20 μM, (b) 200 μM,

and (c) 2 mM (d) 20 mM. In Figure 2-2 (a), the two peaks (540 and 575 nm) in the Q band

region of oxyHb gradually decreased after the addition of 20 μM H2O2 accompanied by

the appearance of a small peak at 630 nm showing an isosbestic point at 586 nm. It is

considered that oxyHb was gradually oxidized to metHb by H2O2 and stopped when H2O2

Chapter 2

- 28 -

Figure 2-2 Spectral changes of the Hb solutions ([heme] = 20 μM) during the reaction with H2O2

at 37 °C ((a) 20 μM, (b) 200 μM, (c) 2 mM, and (d) 20 mM). The repeatative scanning was started

at a 2 min interval, immediately after the addition of a solution of H2O2 to Hb solutions.

Figure 2-3 Spectral changes of the Hb vesicle dispersions ([heme] = 20 μM) during the reaction with

H2O2 at 37 °C ((a) 20 μM, (b) 200 μM, (c) 2 mM, and (d) 20 mM). The repeatative scanning was started

at a 2 min interval, immediately after the addition of a solution of H2O2 to Hb vesicle dispersions.

Chapter 2

- 29 -

was consumed before the completion of the metHb formation. In the dispersion of Hb

vesicles in which a purified Hb solution was encapsulated with a phospholipid bilayer

membrane (particle diameter, 250 nm, Figure 2-3 (a)), the large decline in the baseline

from the lower wavelength typically shows the turbidity of the vesicles. The oxyHb in the

Hb vesicle was converted to metHb after reaction with 20 μM H2O2, and the degree of

metHb conversion was greater than that of the oxyHb solution.

When the added amount of H2O2 was increased to 200 μM, a completely different

feature in the spectral change was observed as shown in Figure 2-2 (b) and 2-3 (b).

Namely, in the Hb solution, the decrease in the two peaks attributed to oxyHb was

accompanied by the total increase in the absorbance from 478 nm to over 700 nm, which

can be identified as ferrylHb. The following growth of the peak at 630 nm then had an

isosbestic point at 612 nm, indicating the conversion of ferrylHb to metHb. A similar trend

was observed for the Hb vesicles except for the baseline change as shown in Figure 2-3 (b).

It is noted that the ratio of metHb in both Hb samples was significantly small after the

reaction with 200 μM H2O2, in comparison with the metHb spectra of the Hb samples

([heme] = 20 μM) completely converted by the reaction with excess NaNO2, suggesting

the existence of discolored Hb products (degraded heme) 21.

When 2 mM and 20 mM of H2O2 were added to the Hb solution, the transient

formation of ferrylHb with the subsequent conversion to the discolored products was

confirmed from the baseline increase with no isosbestic points as shown in Figure 2-2 (c)

and (d). We also confirmed the white aggregates at the bottom of the flask. A similar trend

was also seen in the Hb vesicles but with no aggregation (Figure 2-3 (c) and (d)).

3-3. Changes of Hb components

The composition changes of oxyHb, metHb, ferrylHb, and discolored products

after the addition of H2O2 are summarized in Figure 2-4. These curves were obtained by a

curve fitting technique using computer-simulated spectra from those Hb elements

measured one by one. Figure 2-4 (a) shows the conversion of the oxyHb solution, of which

the heme concentration was 20 μM, to the other Hb products after the reaction with 20 μM

H2O2. The oxyHb was reduced to 66 % within 8 min accompanied by the formation of

29 % metHb. The release of ferric ion and the generation of discolored products were

slightly detected. The amount of H2O2 was below the detectable level (ca. 1 μM) within 7

min, thus showing the catalytic decomposition of H2O2 in this system. The direct

Chapter 2

- 30 -

conversion of oxyHb to metHb induced by a stoichiometric amount of H2O2 was also

reported in some papers 40, 41 and explained in terms of “comproportionation” between

ferrylHb and oxyHb to two metHbs by Giulivi and Davies 41. On the other hand, to the

dispersion of oxyHb vesicles having the heme concentration of 20 μM, the same

concentration of H2O2 was added as shown in Figure 2-4 (b). The oxyHb was reduced to

Figure 2-4 Changes of Hb components and the concentrations of H2O2 in the Hb solution during the

reaction with H2O2 at 37 °C. Hb solutions ([heme] = 20 μM) were reacted with H2O2 ((a) 20 μM, (b)

200 μM and (c) 2 mM, respectively). Each Hb component ratio was calculated from the repeatative

UV-vis spectral changes. (○) oxyHb, (□) metHb, (■) ferrylHb, and (●) discolorated products.

Chapter 2

- 31 -

5 % within 13 min accompanied by the formation of 85 % metHb and 10 % discolored

products. No ferric ion was detected during our observation period (30 min). The main

difference between these two samples is that the conversion of metHb from the oxyHb

solution remained at 29 %, whereas that from the oxyHb vesicle dispersion was over 85 %.

This can be explained as follows. Initially, oxyHb would react with H2O2 to convert

Figure 2-5 Changes of Hb components and the concentrations of H2O2 in the Hb vesicle dispersion

during the reaction with H2O2 at 37 °C. Hb vesicle dispersions ([heme] = 20 μM) were reacted with

H2O2 ((a) 20 μM, (b) 200 μM and (c) 2 mM, respectively). Each Hb component ratio in the Hb

vesicles was calculated from the repeatative UV-vis spectral changes. (○) oxyHb, (□) metHb, (■)

ferrylHb, and (●) discolorated products.

Chapter 2

- 32 -

metHb via comproportionation of ferrylHb with a large amount of oxyHb. The resulting

metHb would show more effective catalase-like reactivity than oxyHb 21, 30. In the Hb

solution, 20 μM H2O2 was completely decomposed by only 6 μM heme. In the case of the

Hb vesicle dispersion, the rate-determining stage would be the reaction between oxyHb

molecules that are located the vicinity of the bilayer membrane inside the vesicle and H2O2

that had just permeated through the bilayer membrane. The resulting metHb would diffuse

toward the core of the vesicle and exchange with unreacted oxyHb, and then the oxyHb

would react with H2O2 to convert metHb. This would result in the higher metHb

conversion and restriction of the catalase-like reaction of metHb. In fact, as shown in

Figure 2-4 (a) and 2-5 (a), the half-lives of the H2O2 decomposition in the Hb solution and

in the Hb vesicle dispersion were 12 s and 115 s, respectively.

The reaction between 10-fold (200 μM) H2O2 and oxyHb (20 μM) in the solution

state or within a vesicle as a dispersion is shown in Figure 2-4(b) and 2-5 (b) as the

conversion of each Hb product. The concentrations of H2O2 and ferric ion are also

included in these figures. OxyHb completely disappeared within 7 min after the addition

of H2O2. It was transiently converted to ferrylHb with a maximum at 4 min, followed by

the conversion of metHb from the ferrylHb. Furthermore, discolored products were

gradually generated from the beginning of the reaction. It is noted that the release of ferric

ion was accompanied by the generation of the discolored products in the oxyHb solution.

However, no ferric ion was observed in the outer aqueous phase of the oxyHb vesicle

dispersion. The half-lives of 200 μM H2O2 in the 20 μM oxyHb solution and the vesicle

dispersion were 21 s and 200 s, respectively. When compared with those in Figure 2-4(a)

and 2-5 (a), only twice the time was needed to decompose the 10-fold H2O2. It is indicated

that the catalase-like activity that cycles between metHb and ferrylHb (globin) radical,

which is one oxidizing equivalent above ferrylHb, remained during the reaction. As

described above, once metHb formed by the reaction of oxyHb with H2O2 via ferrylHb,

the resulting metHb should be rapidly oxidized by H2O2 to the ferrylHb radical. The ferryl

Hb radical is considered to act like the so-called compound I of catalase, which converts

H2O2 to H2O and O2, and back to metHb 30. Since the latter reaction would be slow in

comparison with the ferrylHb formation, the ferrylHb from oxyHb and ferrylHb radical

from metHb are apparently the main products during the reaction with H2O2. These

ferrylHbs gradually returned to metHb after the extinction of H2O2 because of its

instability.

The discolorated products should be degraded to heme fragments and apo-protein

Chapter 2

- 33 -

during the redox cycle between oxyHb and ferrylHb, because we confirmed that metHb

showed a more stable equilibrium state between metHb and the ferrylHb radical during

reaction with H2O2 (data not shown). A recent report described that heme degradation was

caused by superoxide generated from the reaction of ferrylHb with H2O2

21. It is easy to

understand that heme degradation triggered the release of ferric ion, and the resulting

apo-proteins were so unstable that they aggregated as white precipitates. The generation of

such discolored products indicates the non-perfect catalase-like activity of Hb against

H2O2. When 1000-fold H2O2 was added to the Hb sample solutions, oxyHb in both cases

instantly disappeared to convert ferrylHb as an intermediate, and interestingly, the final

product was not metHb but discolored products. This indicates that the

comproportionation of ferrylHb with oxyHb did not occur because almost all of the oxyHb

was instantly converted to ferrylHb, and heme degradation should occur due to the

reaction of ferrylHb with the large amount of H2O2. The decomposition of H2O2 could also

be recognized even if no ferrylHb and metHb existed. The half-lives of H2O2 in the oxyHb

solution and the oxyHb vesicle dispersion were 390 s and 760 s, respectively. H2O2 was

hardly decomposed if H2O2 was added to the discolored Hb products in the presence of

DFO, indicating that the discolored products should have no catalase activity, but H2O2

should be decomposed by a Fenton reaction caused by ferric ion released from the

degraded heme. For the Hb vesicles, we did not observe the ferric ion in the outer aqueous

phase; therefore, H2O2 should be decomposed by ferric ion in the inner aqueous phase of

the Hb vesicle.

4. Lipid peroxidation of egg yolk lecithin by ferrylHb

4-1. Experimental section

The powder of egg yolk lecithin (EYL) was dispersed into pure water (40 mM) and

hydrated at 4 °C for 2 hr under an argon atmosphere. The resulting multilamellar vesicles

were extruded through the membrane filters with the final pore size of 0.22 μm to prepare

the vesicles of which average diameter is 270 + 50 nm. The Hb samples were mixed with

the EYL vesicles (37 mM) and reacted with H2O2 at 37 °C for 30 min, and trichloroacetic

acid (42 mM) was added to precipitate the Hb samples. The supernatant separated from

the Hb samples by centrifugation (12000g, 30 min) was mixed with thiobarbituric acid

Chapter 2

- 34 -

(TBA, 28 mM) and heated for 30 min at 100 °C. Resulting thiobarbituric acid-reacted

substance in the solution was measured with a fluorescence spectrophotometer (Ex: 532

nm, Em: 553 nm).

4-2. Lipid peroxidation of EYL vesicles

In the reaction with egg yolk lecithin (EYL), we could not confirm that either Hb

or H2O2 had peroxidized the EYL comprising vesicles. However, EYL was surely

peroxidized when H2O2 was added to the EYL vesicle dispersion in the presence of 20 μM

ferric ion, indicating that the hydroxyl radical produced from the Fenton reaction between

ferric ion and H2O2 should cause lipid peroxidation. Such lipid peroxidation was inhibited

by the addition of 1.6 mM DFO as a chelator of ferric ion. To the mixture of 37 mM EYL

vesicles, 20 μM Hb, and 1.6 mM DFO were added various concentrations of H2O2, and

the lipid peroxidation value was measured by a TBA method after incubation for 30 min at

37 oC. The closed circle plots in Figure 2-6 show evidence of the lipid peroxidation in the

triadic reaction system of EYL, Hb, and H2O2.

0.5

Hb (DFO free)

H2O2 (mM)

m

a

londia

ldeh

yd

e

(μ

M)

1.0

1.5

2.0

0

2.0

1.0

3.0

4.0

5.0

Hb (DFO)

HbV (DFO free)

HbV (DFO)

0.5

Hb (DFO free)

H2O2 (mM)

m

a

londia

ldeh

yd

e

(μ

M)

1.0

1.5

2.0

0

2.0

1.0

3.0

4.0

5.0

Hb (DFO)

HbV (DFO free)

HbV (DFO)

Figure 2-6 Lipid peroxidation of the EYL vesicles catalyzed by the Hb samples during the reaction

with H2O2 in the presence or absence of DFO. The 37 mM EYL vesicle was dispersed in the Hb

solution or the Hb vesicle dispersion ([heme] = 20 μM)and reacted with H2O2 at 37 °C for 30 min.

Chapter 2

- 35 -

No concentration dependence of H2O2 was seen above the H2O2 concentration of

more than 1.0 mM. This suggests that the rate-determining process would be the reaction

of Hb with EYL because both concentrations were constant. Therefore, we looked at the

relationship between the concentration of ferrylHb and the concentration of

malondialdehyde. The linear relationship proves that the ferrylHb converts EYL to the

peroxidized lipid (data not shown here). To study the influence of the ferric ion released

from denatured Hb, the same experiment was carried out in the absence of DFO. The open

circle plots in Figure 2-6 clearly demonstrate that the ferric ion significantly contributes to

the lipid peroxidation through the Fenton reaction. The proportional relationship between

the ferric ion concentration from FeCl3 artificially added to the solution instead of Hb and

the concentration of the resulting malondialdehyde suggests that the reaction of H2O2 and

ferric ion released from the Hb denatured by H2O2 should produce the hydroxyl radical

which results in the lipid peroxidation. On the other hand, in the Hb vesicle dispersion, the

small increase in malondialdehyde despite the absence of DFO is due to the encapsulation

effect of Hb and ferric ion with the saturated phospholipid bilayer membrane. This

suggests the low cytotoxicity of the endotherial cells in the Hb vesicles in comparison with

Hb modifications under oxidative stress 33. We are now studying the effect of the Hb

samples reacted with H2O2 on the endotherial cells.

5. Supremacy of Hb vesicles over acellular type HBOCs

Figure 2-7 concludes our experimental results. H2O2 reacts with the acellular-type

Hb solution to produce metHb and ferrylHb. The ferrylHb and ferric ion from those

unstable Hb products facilitates the lipid peroxidation of the EYL vesicles, which

represents the cytotoxicity of the reaction series. On the other hand, for the cellular Hb

vesicle dispersion, those products are also generated by the reaction of Hb with H2O2

permeated through the bilayer membrane of the vesicle. However, they are stably

encapsulated within the vesicle and do not cause the lipid peroxidation due to the

separation from the EYL vesicles. These results indicate the high safety of the Hb vesicles

which enclose the reactive Hb products in the reaction with H2O2.

Chapter 2

- 36 -

cellular type (Hb vesicle)

acellular type (Hb)

Hb(Fe2+)

H2O2

H2O2

EYL vesicle

Lipid peroxidation

LH → LOOH

metHb(Fe3+)

ferrylHb(Fe4+=O)

Fe ion

H2O, O2

×

Hb(Fe2+)

H2O2

metHb(Fe3+)

ferrylHb(Fe4+=O)

H2O, O2

Fe ion

cellular type (Hb vesicle)

acellular type (Hb)

Hb(Fe2+)

H2O2

H2O2

H2O2

EYL vesicle

Lipid peroxidation

LH → LOOH

metHb(Fe3+)

ferrylHb(Fe4+=O)

Fe ion

H2O, O2

×

Hb(Fe2+)

H2O2

metHb(Fe3+)

ferrylHb(Fe4+=O)

H2O, O2

Fe ion

Figure 2-7 Concept of the interaction of acellular-type Hb and cellular-type Hb vesicle with H2O2,

and their effects on lipid peroxidation of EYL vesicles.

Chapter 2

- 37 -

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Chapter 3

- 41 -

Chapter 3

Reduction of MetHb in Hb Vesicles

Using Membrane Permeability of Reductants

Contents

1. Introduction

2. Autoxidation mechanism of metHb formation from oxyHb

2-1. autoxidation of oxyHb

2-2. Influence of pO2 to autoxidation

3. MetHb reduction system in red blood cells

3-1. Enzymatic reduction system

3-2. Non-enzymatic reduction system

4. Reduction of metHb by reductants under anaerobic condition

4-1. Experimental section

4-2. Structure of reductants

4-3. UV-vis measurement

5. Reduction of metHb by reductants under aerobic condition

5-1. Experimental section

5-2. UV-vis measurement

6. Autoxidation of reductants and inhibition of metHb reduction

6-1. Experimental section

6-2. Rate constants of autoxidation and metHb reduction of reductants

6-3. Inhibition of metHb reduction

7. Reduction of metHb in Hb vesicles using membrane permeability of reductants

7-1. Experimental section

7-2. MetHb vesicles reduction under anaerobic condition

7-3. Rate constants of metHb reduction in Hb vesicles

7-4. MetHb vesicles reduction under aerobic condition

8. Effect of catalase co-encapsulation on metHb reduction in Hb vesicles

8-1. Experimental section

8-2. Effect of catalase co-encapsulation

9. Kinetics of metHb reduction in Hb vesicles

References

Chapter 3

- 42 -

1. Introduction

MetHb formation of HBOCs under physiologic condition is very important

problem in the development of HBOCs. The heme containing Hb is buried in the

hydrophobic pocket (heme pocket) to prevent the immediate oxidation to ferric state in the

aqueous environment. However, the oxidation by the one electron transfer from ferrous

iron to binding O2 is not avoidable. As a result, metHb and superoxide anion radical (O2

-*)

are produced from oxyHb. It is called “autoxidation” of Hb molecules. MetHb do not have

O2 binding and carrying ability. MetHb is non-functional Hb if it is not reduced to ferrous

state Hb.

In red blood cells, the percentage of metHb is kept under 1 % by the enzymatic

electron control using glucose as a substrate 1. Furthermore, enzymatic systems 2 and

non-enzymatic 3 systems which eliminate reactive oxygen species (ROS) work for the

suppression of metHb formation by ROS, especially H2O2 described in previous chapter.

However, enzymes, low molecular compounds, and other proteins are completely

removed in the process of Hb purification from donated human blood for the Hb vesicle

preparation 4. The purification of Hb solution is up to 99.9 % (36-40 g/dl). Therefore, Hb

vesicles which encapsulate only purified Hb within the lipid bilayer membrane are

non-defensive against ROS, in particular H2O2

5. Acellular-type HBOCS also have the

same problem. Chang et al reported the catalase-SOD conjugated Hb for the artificial

oxygen carrier. Of course, the reason of the conjugation was the suppression of metHb

formation by elimination of ROS using conjugated catalase and SOD. However, the

required catalase and SOD for the significant effect of suppression of metHb formation

were 10 times amount compared with the amount of them in the red blood cells 6.

The problem of metHb formation is had to solve as soon as possible, and it is not

avoidable when Hb is used for artificial oxygen carriers. On the basis of this problem, we

attempted metHb reduction to ferrousHb (oxyHb or deoxyHb) in the Hb vesicles using

membrane permeability of reductants which had low molecular weight. Furthermore, we

used catalase for the H2O2 reduction. We analyzed the kinetics of these reactions for the

construction of metHb reduction system in the Hb vesicles using membrane permeability

of reductants.

Chapter 3

- 43 -

2. Autoxidation mechanism of metHb formation from oxyHb

The main factor of metHb formation in the Hb vesicles was the reaction with ROS,

in particular H2O2, and it was already described in previous chapter. Here, we described

another main factor of metHb formation from oxyHb called autoxidation.

2-1. autoxidation of oxyHb

Autoxidation of oxyHb was considered as the one of chain reaction composed

some elementary reactions. First, metHb and O2

-* were produced from oxyHb (first step),

the O2

-* was dismutated to H2O2 by protonation. The generated H2O2 reacted with oxyHb

to form metHb (second step). Thus, H2O2 is deeply related to autoxidation mechanism,

H2O2 cause the secondary metHb formation following autoxidation 7. The concept of

autoxidation is shown in Figure 3-1.

Jensen et al. reported that ferrous Fe ion of oxyheme was defined as the

pseudo-complex structure of ferric iron heme and O2

-* 8. Therefore, metHb was

generated by the dissociation of O2

-* from oxyHb, in actually, O2

-* generation was

confirmed in the process of Hb autoxidation 7. However, the ∆G of this reaction was +12.7

kcal/mol, it could not be spontaneously proceed. Namely, autoxidation is not simple

reaction that O2

-* is released from oxyHb, It would be obeyed to the mechanism shown in

figure 3-2 advocated by Philips et al 9.

Figure 3-1 Concept of autoxidation of oxyHb in the process of the reversible binding of O2.

Chapter 3

- 44 -

They reported that O2 binding heme in the angle of 121o was stabilized by the hydrogen

bond between binding O2 and distal histidine. As a result, one electron transfer to binding

O2 from ferrous heme iron was facilitated by the polarization of binding O2 (δ-) by the

release of hydrogen-bonded proton of distal histidine with binding O2. The reaction would

be triggered by the intrusion of proton to heme pocket. It was agreed to the fact that metHb

formation of oxyHb was facilitated under the lower pH condition. It is called proton

oxidation of ferrous heme.

2-2. Influence of pO2 to autoxidation

Autoxidation rate of oxyHb which P50 of the is controlled at 30 Torr (the same

value with Hb in human red blood cells) increases following the decrease of pO2, and the

rate shows maximum value at the same point of P50 (30 Torr) 10. Under the lower pO2 than

30 Torr, the rate gradually decreases following pO2 decrease. Figure 3-3 is shown the

relationship of autoxidation rate and pO2.

Figure 3-2 O2 binding state and autoxidation mechanism of oxyHb advocated by Philips et al.

Figure 3-3 Relationship of autoxidation rate and pO2. It showed the

maximal value at the 30 Torr (P50 of this Hb)

Chapter 3

- 45 -

3. MetHb reduction system in red blood cells

3-1. Enzymatic reduction system

NADH-cytochrome b5 reductase system 2

In 1971, metHb reduction by NADH-cytochrome b5 system in red blood cell was

clarified by the discovery of cytochrome b5 in red blood cells by Juckett et al 11. The

mechanism is shown in Figure 3-4.

MetHb is effectively reduced to ferrousHb by the cytochrome b5 as an agency.

Cytochrome b5 exists only 0.2 – 1 μM in red blood cells. The reduced Cytochrome b5

(Fe2+) generated by the reaction with NADH-cytochrome b5 reductase system reduces

metHb to ferrousHb by the non-enzymatic reaction. The non-enzymatic reduction

occurred only by the redox potentials of metHb and reduced cytochrome b5. However,

enzymatic reduction by NADH-cytochrome b5 reductase is absolutely required in the

reduction of ferric to ferrous cytochrome b5. In the cycle shown in Figure 3-4, the

rate-determining step is non-enzymatic reduction.

NADH-flavin reductase system 12

MetHb reduction by NADH-flavin reductase system is shown in figure 3-5. The

reduction is separated into two steps. 1) Enzymatic flavin reduction by NADH-flavin

reductase, 2) Non-enzymatic metHb reduction to ferrousHb by reduced flavin. It is well

known that the flavin reduction by the NADH-flavin reductase is not so fast, however,

Figure 3-4 MetHb reduction by NADH-cytochrome b5 reductase system

in red blood cells.

cytochrome b5

(Fe2+)

NAD

NADH

cytochrome b5

(Fe3+)

metHb (Fe3+)

ferrousHb (Fe2+)

metHb

formation

NADH-cytochrome b5

reductase

Non-enzymatic

reduciton

cytochrome b5

(Fe2+)

NAD

NADH

cytochrome b5

(Fe3+)

metHb (Fe3+)

ferrousHb (Fe2+)

metHb

formation

NADH-cytochrome b5

reductase

Non-enzymatic

reduciton

Chapter 3

- 46 -

metHb reduction to ferrousHb is quite fast. Therefore, the rate determining step does not

exist in the reduction system.

3-2. Non-enzymatic reduction system 3

The main compounds of non-enzymatic reduction system of metHb to ferrousHb

in red blood bells are considered to be ascorbic acid (AsA) and glutathione (GSH). Even if

it is the non-enzymatic system, enzymatic systems work for the recycle of oxidized

substrates generated by the metHb reduction to ferrousHb. AsA is oxidized to

dehydro-AsA (oxidized AsA) by the reduction of metHb. The dehydro-AsA is considered

to be re-reduced to AsA by dehydroascorbic acid reductase using glutathione as an agency.

The concept is shown in Figure 3-6.

Figure 3-5 MetHb reduction by NADPH-flavin reductase system in red blood cells.

Figure 3-6 Non-enzymatic metHb reduction system working AsA and GSH in red blood cells.

flavin (oxidized)

NADPH

NADP+

metHb (Fe3+)

ferrousHb (Fe2+)

NADPH-flavin

reductase

Non-enzymatic

reduction

flavin (reduced)

flavin (oxidized)

NADPH

NADP+

metHb (Fe3+)

ferrousHb (Fe2+)

NADPH-flavin

reductase

Non-enzymatic

reduction

flavin (reduced)

glutathione

(oxidized)

NAD(P)H

NADP

Glutathione

(reduced)

AsA (reduced)

Dehydro-AsA

(oxidized)

glutathione reductase Dehydroascorbic acid

reductase

metHb (Fe3+)

ferrousHb (Fe2+)

Non-enzymatic

reduction

glutathione

(oxidized)

NAD(P)H

NADP

Glutathione

(reduced)

AsA (reduced)

Dehydro-AsA

(oxidized)

glutathione reductase Dehydroascorbic acid

reductase

metHb (Fe3+)

ferrousHb (Fe2+)

Non-enzymatic

reduction

Chapter 3

- 47 -

4. Reduction of metHb by reductants under anaerobic condition

4-1. Experimental section 13

MetHb was prepared by the reaction of HbCO (10 g/dl) with 2.5-fold excess

potassium ferricyanide in the heme base. The unreacted potassium ferricyanide and

ferrocyanide were removed by gel permeation chromatography (GPC, Sephadex-G25)

developed by PBS (pH7.4). Various concentrations of Reductants solution (L-cysteine

(L-Cys), L-homocysteine (L-Hcy), AsA, dithiothreitol (DTT), N-acetylcysteine (Ncy))

were added to the metHb solution ([metHb] = 5 μM, PBS (pH7.4)) under anaerobic

condition (pure nitrogen condition). The reaction was monitored by the repetitive scanning

of a visible region from 300 to 700 nm at 2 min interval by using an UV-vis spectrometry.

4-2. Structure of reductants

First, the structures of thiol compounds used for metHb reduction are shown in

Figure 3-7. In human reds blood cells, thiol compounds and AsA are important for the

metHb reduction system. From the reason, we focused on these compounds. They were

considered to be available for clinical trial. In particular, GSH and AsA are considered to

be contributed to the direct reduction of metHb to ferrousHb in the red blood cells as one

of the non-enzymatic reduction systems.

Figure 3-7 Structures of reductants used for metHb reduction.

L-cysteine (L-Cys)

L-homocysteine (L-Hcy)

dithiothreitol (DTT)

N-acetylcysteine (Ncy)

glutathione (GSH)

L-cysteine (L-Cys)

L-homocysteine (L-Hcy)

dithiothreitol (DTT)

N-acetylcysteine (Ncy)

glutathione (GSH)

Chapter 3

- 48 -

4-3. UV-vis measurement

Figure 3-8 shows the reduction process of metHb to deoxyHb by L-Cys under

anaerobic condition. MetHb was gradually reduced to ferrousHb (deoxyHb) under

anaerobic condition. MetHb was perfectly reduced to deoxyHb after 120 min.

All reductants (L-Cys, L-Hcy, Ncy, AsA, GSH, Ncy) showed the metHb reduction.

However, the denaturation of Hb was confirmed when DTT was used for the reductant. It

was considered that DTT was too strong reductive activity to use for metHb reduction. Hb

molecule has some reduced SH groups come from cysteine of peptide (globin) chains. It

would be considered that DTT caused the reduction and thiol changes of these

intermolecular SH groups of Hb sample. As a result, Hb structure might be changed by

thiol modifications, and the Hb lost the heme holding ability in the heme pocket. Finally, it

leaded up to protein denaturation following heme release.

In particular, Cys and AsA were shown very good profiles of metHb reduction to

deoxyHb in this condition (anaerobic condition). Cys and AsA were considered as

possible and important candidates for the metHb reduction in Hb vesicles at the point in

time. Rate constant of metHb reduction and related constants were described in the later

section.

Wavelength (nm)

1.0

400

Abs

(-)

500

600

700

300

1.5

2.0

2.5

0.5

0

0

10

30

60

120 min

X 5

Wavelength (nm)

1.0

400

Abs

(-)

500

600

700

300

1.5

2.0

2.5

0.5

0

0

10

30

60

120 min

0

10

30

60

120 min

X 5

Figure 3-8 Reduction of metHb (5 μM) to deoxyHb by reductant

(L-Cys (10 mM)) under anaerobic condition.

Chapter 3

- 49 -

5. Reduction of metHb by reductants under aerobic condition

5-1. Experimental section

The experiments in this section were performed the same method described in

previous section (4-1). The difference point from the experiments of previous section was

that experiments were performed “under aerobic condition (pO2 = 40 and 149 Torr)”.

5-2. UV-vis measurement

Figure 3-9 shows the reduction process of metHb to oxyHb by L-Cys under pO2 of

40 Torr. In this case, if metHb was reduced to ferrousHb, it was detected as oxyHb

because of O2 existence. When the pO2 is approximately the P50 of Hb, the rate of metHb

formation tends to show a maximum, and the average oxygen partial pressure in mixed

venous blood is estimated to be 40 Torr. Therefore, we used the constant oxygen partial

pressure of 40 Torr to measure the metHb formation by the reduction for estimation of the

in vivo behavior. MetHb was not reduced to oxyHb by L-Cys or other all reductants. In

addition, denaturation of Hb was detected from the increase of the absorbance at 700 nm

and decrease of 405 nm.

Wavelength (nm)

1.0

400

Abs (-)

500

600

700

300

1.5

2.0

2.5

0.5

0

0

10

30

60

120 min

Wavelength (nm)

1.0

400

Abs (-)

500

600

700

300

1.5

2.0

2.5

0.5

0

0

10

30

60

120 min

0

10

30

60

120 min

Figure 3-9 Reduction of metHb (5 μM) to deoxyHb by reductant

(L-Cys (10 mM)) under pO2 of 40 Torr.

Chapter 3

- 50 -

The same result was obtained under the pO2 of 149 Torr (under air). The

reductions of various reductants were surely proceeded under anaerobic condition (pure

nitrogen, pO2 = 0 Torr) from the results of previous section. However, they were not

proceeded in the presence of O2. I was considered that the O2 was reduced by the

reductants. It was called autoxidation of reductants. The autoxidation rate constant might

be larger then metHb reduction rate constants by these reductants. If the idea was current,

autoxidation inhibited the metHb reduction. The detail verification and discussion about

autoxidation were described in the next section.

6. Autoxidation of reductants and inhibition of metHb reduction

6-1. Experimental section

Autoxidation rate of reductants were measured using the thiol exchanging reaction

between reductants and 2,2-ditiodipyridine. The mechanism is shown in Figure 3-10. Each

reductant solution in PBS (pH7.4) was incubated and stirred at 37 oC under the pO2 of 149

Torr (under air). They were periodically sampled out and immediately mixed with

2,2-ditiodipyridine (2.5 mM). The mixed samples were measured by UV-vis spectrometry

(300 – 700 nm). The measurements were performed using various concentrations of

reductants for the calculating autoxidation rate constants. The thiol concentrations were

calculated from the molal absorptivity at 343 nm (ε = 7060) of formed 2,2-ditiodipyridine

derivatives.

6-2. Rate constants of autoxidation and metHb reduction of reductants

Table 3-1 shows the autoxidation rate constants of reductants under pO2 of 149

Torr (under air) and the metHb reduction rate constants of reductants under anaerobic

condition (pO2 = 0 Torr).

2,2-dithiodipyridine

2,2-dithiodipyridine derivative

(fluorescent compound)

2,2-dithiodipyridine

2,2-dithiodipyridine derivative

(fluorescent compound)

Figure 3-10 Thiol exchanging reaction of 2,2-ditiodipyridine and mechanism of thiol assay method.

Chapter 3

- 51 -

The detail discussion of the results was described in the next.

6-3. Inhibition of metHb reduction

In autoxidation, O2 is reduced by the electron donation from reductants. It is well

known that O2 molecule reduced by the reductants forms superoxide anion radical (O2

-*).

It is one of the ROS. The mechanism is shown in Figure 3-11.

In the experiments under aerobic conditions, It was considered that the generated

O2

-* was dismutated to H2O2, and the reduced ferrousHb by reductants was attacked by

the formed H2O2. All reductants had the larger rate constants of autoxidation than those of

metHb reduction shown in Table 3-1. Namely, all reductants had the relationship of

following inequality.

kox

> kred

Table 3-1 Autoxidation rate constants and metHb reduction rate constants

of reductants. Measurements of the autoxidation and the reduction were

measured under pO2 of 149 Torr and 0 Torr, respectively.

2RSH

O

2

O

2

-*

RSSR

metHb (Fe3+)

ferrousHb (Fe2+)

kox

kred

2RSH

O

2

O

2

-*

RSSR

metHb (Fe3+)

ferrousHb (Fe2+)

kox

kred

Figure 3-11 Autoxidation and metHb reduction mechanisms of thiol compounds.

8.15

reductants

pKa

metHb reduction rate

constant kred (M-1s-1)

thiol autoxidation rate

constant kox (M-1s-1)

L-Cys

L-Hcy

DTT

GSH

Ncy

1.7 x 10-2

8.70

8.56

9.52

-

1.2 x 10-2

7.0 x 101

2.5 x 10-3

1.8 x 10-3

7.5 x 10-2

2.8 x 10-2

6.2 x 10-2

1.2 x 10-2

-

8.15

reductants

pKa

metHb reduction rate

constant kred (M-1s-1)

thiol autoxidation rate

constant kox (M-1s-1)

L-Cys

L-Hcy

DTT

GSH

Ncy

1.7 x 10-2

8.70

8.56

9.52

-

1.2 x 10-2

7.0 x 101

2.5 x 10-3

1.8 x 10-3

7.5 x 10-2

2.8 x 10-2

6.2 x 10-2

1.2 x 10-2

-

Chapter 3

- 52 -

It was considered that the total amount of generated H2O2 was larger than that of

reduced ferrousHb from metHb by reductants, and the reduced ferrousHb was

immediately oxidized to metHb by the reaction with H2O2 generated by the autoxidation

of reductants. As a result, only metHb was detected in the UV-vis measurement. It would

be considered that the detection was related to the scanning speed of UV-vis spectrometry,

and the scanning speed of UV-vis spectrometry was too late to detect ferrousHb (oxyHb).

If the measurement was performed by difference methods (e.g. stopped-flow measurement,

etc.), the ferrous state might be detected.

From these results, It was clarified that metHb reduction by reductants was not

available under aerobic condition (pO2 = 40 Torr and 149 Torr). However, Hb molecules

in the Hb vesicles existed under the quite different environment compared with Hb

solution used in these experiments. The reaction field might be difference from these

experiments. For example, the Hb concentration in the Hb vesicle (reaction field) was 22.3

mM (36 g/dl). It was more than 4000 times value compared with the Hb solution (5 μM)

used in this section. From these considerations, we challenged metHb reduction in the Hb

vesicles using membrane permeability of reductants.

7. Reduction of metHb in Hb vesicles using membrane permeability of reductants

7-1. Experimental section

Preparation of metHb vesicles

Pyridoxal 5’-phosphate (PLP) was added to the Hb solution as an allosteric

effector at a 2.5 equimolar ratio of PLP to HbCO (36 g/dl). The pH of the solution was

adjusted to 7.4 by 0.2 N NaOH. To prepare the Hb vesicles, mixed lipid powders (DPPC /

cholesterol / DPEA/ PEG-DSPE = 5 / 5 / 1 / 0.033) was added to the HbCO solution and

the mixture was stirred at 25 oC for 12 hr. The resulting dispersion of the multilamellar

vesicles was subsequently extruded through the nitrocellulose membrane filters with a

pore size of 0.22 μm (Fuji Film Co., Tokyo) to prepare the Hb vesicles with an average

diameter of 253 ± 35 nm. After the separation of unencapsulated Hb by ultracentrifugation

(10000g, 60 min), the precipitate of the Hb vesicles was redispersed into saline in order to

adjust the Hb concentration of the Hb vesicle dispersion to 10 g/dl. HbCO within the

vesicles was decarbonyzed and oxygenated to HbO2 by irradiation of visible light onto a

Chapter 3

- 53 -

liquid film under O2 atmosphere. MetHb vesicles were prepared by the reaction of Hb

vesicles (10 g/dl) with an excess amount of sodium nitrite as an oxidant. The amount of

oxidant was for the complete Hb oxidation of ferrousHb (HbO2) to metHb in the Hb

vesicles. The unreacted sodium nitrite was perfectly removed by gel permeation

chromatography (GPC, Sephadex G-25).

Reduction of metHb vesicles by reductants

Various concentrations of Reductants solution (L-Cys, L-Hcy, AsA) were added to

the metHb vesicle dispersion ([metHb] = 5 μM, PBS (pH7.4)) under anaerobic and aerobic

condition (pO2 = 0, 40, and 149 Torr). The reaction was monitored by the repetitive

scanning of a visible region from 300 to 700 nm at 2 min interval by using an UV-vis

spectrometry.

7-2. MetHb vesicles reduction under anaerobic condition

From the results of previous section, Cys, L-Hcy, and AsA were recommended as

the candidates for the reductants of metHb vesicle reduction. They were selected on the

basis of total judgements of effect, autoxidation, and safety. In this experiment, all Hb

molecules in the Hb vesicles were metHb.

Figure 3-12 shows the reduction process of metHb in Hb vesicles to deoxyHb by

L-Cys under anaerobic condition. MetHb in Hb vesicles was gradually reduced to

ferrousHb (deoxyHb) under anaerobic condition. MetHb in Hb vesicles was perfectly

reduced to deoxyHb after 180 min. it was almost same profile compared with metHb

solution. From the result, it indicated that L-Cys permeated through lipid bilayer

membrane of Hb vesicles composed from mixed lipid (DPPC / cholesterol / DPEA /

PEG-DSPE) and reduced metHb to deoxyHb in Hb vesicles. Namely, L-Cys had

membrane permeability and metHb reduction ability also in Hb vesicles. L-Hcy also

showed the metHb reduction in Hb vesicles. It meant that L-Hcy also had membrane

permeability and metHb reduction ability in Hb vesicles. However, the reduction rate

constant was smaller than that of L-Cys. The details reduction rate constants were

described in the next section. On the other hand, metHb in Hb vesicle was not reduced by

AsA at all. It would be considered that AsA could not permeate through the membrane.

Chapter 3

- 54 -

7-3. Rate constants of metHb reduction in Hb vesicles

Rate constants of metHb reduction in Hb vesicles by the membrane permeation of

reductants are shown in Table 3-2.

Reductants showed smaller metHb reduction rate constants in Hb vesicles

compared with metHb solution. It was considered that there was a lipid bilayer membrane

as an obstruction in the case of Hb vesicles. Reductants had to permeate the membrane for

the metHb reduction in Hb vesicles, it resulted to the differences of rate constants between

metHb solution and metHb vesicles. Furthermore, molecular sizes and charges of

reductants would be related to membrane permeability and metHb reduction rate constant.

In particular, one of OH groups of AsA (pKa = 5.34) was dissociated to O- state in the PBS

Figure 3-12 Reduction of metHb in Hb vesicles (5 μM) to deoxyHb

by reductant (L-Cys (10 mM)) under anaerobic condition.

700

Wavelength (nm)

1.0

400

A

b

s (-)

500

600

300

1.5

2.0

2.5

0.5

0

0

15

30

60

120

X 3

3.0

180 min

700

Wavelength (nm)

1.0

400

A

b

s (-)

500

600

300

1.5

2.0

2.5

0.5

0

0

15

30

60

120

X 3

3.0

180 min

Table 3-2 Autoxidation rate constants and apparent metHb reduction rate

constants in Hb vesicles of reductants. Measurements of the autoxidation and the

reduction were measured under pO2 of 149 Torr and 0 Torr, respectively.

8.15

reductants

pKa

metHb reduction rate

constant kred (M-1s-1)

autoxidation rate

constant kox (M-1s-1)

L-Cys

L-Hcy

AsA

7.0 x 10-3

8.70

5.34

3.2 x 10-3

Not reduced

7.5 x 10-2

2.8 x 10-2

-

8.15

reductants

pKa

metHb reduction rate

constant kred (M-1s-1)

autoxidation rate

constant kox (M-1s-1)

L-Cys

L-Hcy

AsA

7.0 x 10-3

8.70

5.34

3.2 x 10-3

Not reduced

7.5 x 10-2

2.8 x 10-2

-

Chapter 3

- 55 -

(pH7.4) solution. Lipid bilayer membrane composing Hb vesicle did not permeate almost

anionic compounds in spite of their sizes 14. It was applicable to O2

-* and hydroxyl radical

(OH-*). Unfortunately, H2O2 could permeate through the membrane. From our studies,

permeability of H2O2 was very high, and it seemed that the membrane did not play the rule

as an obstruction.

7-4. MetHb vesicles reduction under aerobic condition

Figure 3-13 shows the reduction process of metHb to oxyHb in Hb vesicles by

L-Cys under pO2 of 40 Torr. In this case, if metHb was reduced to ferrousHb, it was

detected as oxyHb because of O2 existence. MetHb in Hb vesicles was not reduced to

oxyHb at all by L-Cys, L-Hcy, or AsA. However, the turbidity increase at 700 nm was not

detected, the denaturation of Hb would not formed outside of Hb vesicles. It might be

formed in Hb vesicles, however, staying denaturation products or released heme iron from

Hb was one of the merits of Hb vesicles already described in Chapter 2.

The same result was obtained under the pO2 of 149 Torr (under air). The

reductions of L-Cys and L-Hcy were surely proceeded under anaerobic condition (pure

nitrogen, pO2 = 0 Torr) from the results of previous section. They were not also proceeded

700

Wavelength (nm)

1.0

400

A

b

s (-)

500

600

300

1.5

2.0

2.5

0.5

0

3.0

0

15

30

60

120

180 min

700

Wavelength (nm)

1.0

400

A

b

s (-)

500

600

300

1.5

2.0

2.5

0.5

0

3.0

0

15

30

60

120

180 min

Figure 3-13 Reduction of metHb in Hb vesicles (5 μM) to deoxyHb

by reductant (L-Cys (10 mM)) under pO2 of 40 Torr.

Chapter 3

- 56 -

in the presence of O2 in the case of metHb solution. It was also the influence of

autoxidation of reductants shown in Table 3-2. Both L-Cys and L-Hcy had larger rate

constants of autoxidation than those of metHb reduction. From these results, even if the

lipid bilayer membrane existed, the reduction profiles of metHb to ferrousHb (deoxy- or

oxyHb) was correspondence each other. Only the reduction rate constants was difference

between metHb solution and metHb in Hb vesicles dispersion by the existence of

membrane as an obstruction of permeation. Figure 3-14 shows the concept of inhibition of

metHb reduction in Hb vesicles.

8. Effect of catalase co-encapsulation on metHb reduction in Hb vesicles

8-1. Experimental section

Preparation of catalase co-encapsulating metHb vesicles

Pyridoxal 5’-phosphate (PLP) was added to the Hb solution as an allosteric

effector at a 2.5 equimolar ratio of PLP to HbCO (36 g/dl). Catalase (final concentration;

2Cys

autoxidation

O2

O2-*

dismutation

Cys-S-S-Cys

H2O2

Cys

metHb

reduction

oxyHb

metHb

Figure 3-14 Concept of metHb reduction using membrane permeability of reductant

(L-Cys) and its inhibition by autoxidation of the reductant.

Chapter 3

- 57 -

2.0 x 105 unit/mL) was added to the HbCO solution. The preparation scheme after catalase

addition was the same as the scheme described in section 7-1 in this chapter.

Reduction of catalase-coencapsulating metHb vesicles by reductants

Various concentrations of L-Cys solutions were added to the metHb vesicle

dispersion ([metHb] = 2 g/dl, PBS (pH7.4)) under aerobic condition (pO2 = 40, and 149

Torr). The reaction was monitored by the repetitive scanning of a visible region from 300

to 700 nm at 2 min interval by using an UV-vis spectrometry. The percentages of metHb

reduction were calculated from the results of UV-vis spectrometry.

8-2. Effect of catalase co-encapsulation

Figure 3-15 shows the metHb reduction to oxyHb in catalase co-encapsulating Hb

vesicles (catalase; 5.0 x 104 unit/ml) by L-Cys under pO2 of 149 Torr (under air).

It was confirmed that metHb was a little reduced to oxyHb in catalase

co-encapsulating Hb vesicles. The result was our first achievement of metHb reduction in

Hb vesicles under aerobic condition (air condition). H2O2 should be generated by the

autoxidation of L-Cys under this experimental condition. It was considered that a part of

generated H2O2 was eliminated by co-encapsulating catalase, as a result, metHb reduction

was confirmed by UV-vis spectrometry. Then, we tried this experiment under pO2 of 40

Torr. The result is shown in Figure 3-16.

50

Time (min)

0

10

20

30

40

60

20

40

60

80

100

2 g/dl HbV + 10 mM L-Cys

2 g/dl HbV + 50 mM L-Cys

me

tH

b

in

H

b

V

(%

)

pO2 = 149 Torr

50

Time (min)

0

10

20

30

40

60

20

40

60

80

100

2 g/dl HbV + 10 mM L-Cys

2 g/dl HbV + 50 mM L-Cys

me

tH

b

in

H

b

V

(%

)

pO2 = 149 Torr

Figure 3-15 Reduction of metHb in catalase co-encapsulating Hb

vesicles (2 g/dl) to oxyHb by reductant (L-Cys) under pO2 of 149 Torr.

Chapter 3

- 58 -

In the experiment of L-Cys concentration of 50 mM, a remarkable reduction of metHb to

oxyHb in catalase co-encapsulating Hb vesicles (catalase; 5.0 x 104 unit/ml) was

confirmed. Autoxidation rate of L-Cys was decreased following pO2 decrease. On the

other hand, metHb reduction rate by L-Cys would not be change even if the pO2 was

changed. Namely, the generation of H2O2 was suppressed compared with pO2 of 149 Torr,

as a result, it was considered that the reduction of metHb to oxyHb by L-Cys in Hb

vesicles effectively proceeded.

9. Kinetics of metHb reduction in Hb vesicles

For the application of kinetics in dilute Hb vesicle dispersion (5 μM) to

concentrated Hb vesicle dispersion (over 1 g/dl (155 μM)), we tired the metHb reduction

in Hb vesicles by L-Cys in the concentrated Hb vesicle dispersion under anaerobic

condition (pure nitrogen, pO2 = 0 Torr). The results are shown in Figure 3-17. The 1 g/dl

of Hb vesicle was equal to 155 μM of Hb concentration.

We confirmed that metHb in vesicles was reduced by the addition of L-Cys. In 5

g/dl metHb vesicle dispersion, with increase of L-Cys concentration from 20 to 50 mM,

the reduction rate was increased as shown in Figure 3-17. And at the addition of 50 mM

L-Cys, the reduction rate was increased with increase of the concentration of Hb vesicle

from 1.0 to 5 g/dl. From these results, the relationships of metHb, met-heme and L-Cys of

the reduction shown as following equations;

75

Time (min)

0

15

30

45

60

90

20

40

60

80

100

2 g/dl HbV + 10 mM L-Cys

2 g/dl HbV + 50 mM L-Cys

me

tH

b in

H

b

V

(%

)

pO2 = 40 Torr

75

Time (min)

0

15

30

45

60

90

20

40

60

80

100

2 g/dl HbV + 10 mM L-Cys

2 g/dl HbV + 50 mM L-Cys

me

tH

b in

H

b

V

(%

)

pO2 = 40 Torr

Figure 3-16 Reduction of metHb in catalase co-encapsulating Hb

vesicles (2 g/dl) to oxyHb by reductant (L-Cys) under pO2 of 40 Torr.

Chapter 3

- 59 -

)(2

　

　

)

[Cys]

([Cys]

dt

d[Cys]

　

.......

　

　　

　

　　　　

i

e

2

i

　　

−

= k

where, k1 is heme(Fe3+) reduction rate constant (M-1s-1), A is coefficient about total surface

area of Hb vesicles (-)),

where, k2 is membrane permeation rate constant (s-1).

We calculated [heme(Fe3+)]i = 2.5x10-2 (M), k1 = 6.7x10-2 (M-1s-1), and A = 3.9x10-2

(M). [heme(Fe3+)]i is a heme concentration in a vesicle, [Cys]e, and [Cys]i are L-Cys

concentration at exterior and interior of a vesicle, respectively. These formulas indicated

that Hb vesicle (5 g/dl) when 20 % metHb was contained, was completely reduced within

30 min by the infusion of a L-Cys solution (50 mM) under pO2 of 40 Torr in vivo.

Figure 3-17 Reduction of metHb to deoxyHb in concentrated Hb vesicles

by reductant (L-Cys) under anaerobic condition (pO2 = 0 Torr). ○ 5g/dl

metHb Vesicles + 50 mM L-Cys ● 5g/dl metHb Vesicles + 40 mM L-Cys

□ 5g/dl metHb Vesicles + 20 mM L-Cys ■ 2.5g/dl metHb Vesicles + 50

mM L-Cys △1g/dl metHb Vesicles + 50 mM L-Cys

(1)

[heme]

A

[Cys]

)]3

[heme(Fe

dt

d[metHb]

　

........

i

i

1

×

×

+

=

−

k

0

40

deoxyH

b (μ

M)

20

60

80

100

200

300

400

100

0

40

deoxyH

b (μ

M)

20

60

80

100

200

300

400

100

Chapter 3

- 60 -

References

1. Srivastava, S., Alhomida, A. S., Siddiqi, N. J., Pandey, V. C., and Puri, S. K., Effect

of beta-arteether treatment on erythrocytic methemoglobin reductase system in

Plasmodium yoelii nigeriensis infected mice, Drug Chem. Toxicol. 24 (2001)

181-190.

2. Zerez, C. R., Lachant, N. A., and Tanaka, K. R., Impaired erythrocyte methemoglobin

reduction in sickle cell disease: dependence of methemoglobin reduction on reduced

nicotinamide adenine dinucleotide content, Blood 76 (1990) 1008-1014.

3. Mawatari, S., and Murakami, K., Different types of glutathionylation of hemoglobin

can exist in intact erythrocytes, Arch. Biochem. Biophys. 421 (2004) 108-114.

4. Sakai, H., Tomiyama, K., Sou, K., Takeoka, S., and Tsuchida, E,

Poly(ethyleneglycol)-conjugation and deoxygenation enable long term preservation of

hemoglobin vesicles as oxygen carriers, Bioconjugate Chem. 11 (2000) 425-432.

5. Takeoka, S., Teramura, Y., Atoji, T., and Tsuchida, E., Effect of Hb-encapsulation

with vesicles on H2O2 reaction and lipid peroxidation, Bioconjugate Chem. 13 (2002)

1302-1308.

6. Chang, T. M. S., Blood Substitutes: Principles, Methods, Products and Clinical Trials,

S. Karger AG, Switzerland (1997).

7. Yusa, K., and Shikama, K., Oxidation of oxymyoglobin to metmyoglobin with

hydrogen peroxide: involvement of ferryl intermediate, Biochemistry 26 (1987)

6684-6688.

8. Jensen, K. P., Roos, B. O., and Ryde, U., O2-binding to heme: electronic structure and

spectrum of oxyheme, studied by multiconfigurational methods, J. of Inorg. Biochem.

99 (2005) 45-54.

9. Rosenthal P. J., and Meshnick, S. R., Hemoglobin catabolism and iron utilization by

malaria parasites, Mol. Biochem. Parasitol. 83 (1996) 131-139.

10. H. Sakai, H., Masada, Y., Takeoka, S., and Tsuchida, E., Characteristics of bovine

hemoglobin as a potential source of hemoglobin-vesicles for an artificial oxygen

carrier, J. Biochem. 131 (2002) 611-617.

11. Juckett, D. A., and Hultquist, D. E., Magnetic circular dichroism studies of

hemoglobin: The reduction of ferrihemoglobin by ferrocytochrome b5

and

characterization of the high-spin hydroxy species of mixed-valence hemoglobin,

Biophys. Chem. 19 (1984) 321-335.

Chapter 3

- 61 -

12. Yubisui, T., Matsuki, T., Tanishima, K., Takeshita, M., and Yoneyama, Y., NADPH-

flavin reductase in human erythrocytes and the reduction of methemoglobin through

flavin by the enzyme, Biochem. Biophys. Res. Commun. 76 (1977) 174-182.

13. Takeoka, S, Sakai, H., Kose, T., Mano, Y., Seino, Y., Nishide, H., and Tsuchida E.,

Methemoglobin Formation in Hemoglobin Vesicles and Reduction by Encapsulated

Thiols, Bioconjugate Chem. 8 (1997) 539-544.

14. Teramura, Y., Kanazawa, H., Sakai, H., Takeoka, S., and Tsuchida. E., Prolonged

Oxygen-Carrying Ability of Hemoglobin Vesicles by Coencapsulation of Catalase in

Vivo, Bioconjugate Chem. 14 (2003) 1171-1176.

Chapter 3

- 62 -

Chapter 4

- 63 -

Chapter 4

Construction of H2O2 Elimination System

Using Peroxidase Activity of MetHb

Contents

1. Introduction

2. Reaction of metHb with H2O2 and generation of ferrylHb radical

2-1. Experimental section

2-2. Elimination of H2O2 by the metHb

2-3. UV-vis measurement

3. Preparation of ferrylHb radical and its radical mechanisms

3-1. Experimental section

3-2. UV-vis measurement

3-3. ESR measurement

3-4. Mechanisms of generation, isolation and disappearance of ferrylHb radical

4. Temperature stability of ferrylHb radical

4-1. Experimental section

4-2. Conversion of ferrylHb radical

4-3. Autoreduction of ferrylHb radical

5. Reduction of ferrylHb radical by L-Tyr

5-1. Experimental section

5-2. Reduction of ferrylHb radical by L-Tyr (UV-vis measurement)

5-3. Reduction of ferrylHb radical by L-Tyr (ESR measurement)

5-4. Radical mechanism of ferrylHb radical

6. Analysis of L-Tyr in the reaction of metHb with H2O2

6-1. Experimental section

6-2. HPLC analysis of L-Tyr

6-3. Mechanism of diTyr generation

References

Chapter 4

- 64 -

1. Introduction

Heme-containing proteins show high redox activities in the presence of reactive

oxygen species (ROS). The reaction mechanism has been studied by many investigators in

various fields. It is well known that methemoglobin (metHb, Hb(Fe3+)), which has a ferric

heme iron, is oxidized to ferrylHb radical (Hb(Fe4+=O*)) by reaction with hydrogen

peroxide (H2O2). Malencik et al. reported that horseradish peroxidase (HRP), which is also

a kind of heme-protein, produced an L-Tyr dimer (diTyr) by coupling the ortho-positions

of two L-Tyr molecules as substrate utilizing H2O2

1. This reaction was based on the

generation of the ferryl radical of HRP (HRP(Fe4+=O*), Compound I) by the oxidation of

the ferric state (HRP(Fe3+)) by H2O2 followed by reduction of the radical state to

regenerate the ferric state by the oxidative coupling of L-Tyr to diTyr. This is the

enzymatic cycle of HRP.

The ferryl radical states (Compound I) of Hb and myoglobin (Mb), which are

mainly induced by the reaction with H2O2, are known to be strong oxidants for several

biomolecules such as tocopherol (and its analog Trolox C) 2, 3 and ascorbic acid 4, 5, and are

reduced to the respective ferric states (metHb and metMb) by the acceptance of two

electrons from these biomolecules. DiTyr and its related products were detected in human

erythrocytes and were thought to be produced by the proteolysis of the ferrylHb radical

generated by H2O2

6. Thus, the ferryl radicals of Hb or Mb are unstable due to their high

reactivity. Thus, the ferryl radicals of Hb or Mb are unstable due to their high reactivity.

The enzymatic pseudo-catalase activity is generally shown as follows;

Hb(Fe3+) + H2O2 → Hb(Fe4+=O*) + H2O (1)

Hb(Fe4+=O*) + H2O2→ Hb(He3+) + H2O + O2

(2a)

Hb(Fe4+=O*) → denaturation, heme release (2b)

Because ferrylHb radical is very unstable, therefore, equation (2b) is mainly occurred in

the reaction. This is the reason of that metHb is not worked as H2O2 elimination enzyme

like catalase.Thus, the ferryl radicals of Hb or Mb are unstable due to their high radical

reactivity. Nagababu et al. reported a reaction intermediate generated from the oxoferryl

Chapter 4

- 65 -

state of Hb (Hb(Fe4+=O)) in the process of heme degradation and the proteolysis of Hb by

H2O2, and named it “rhombic heme” 7.

We have developed Hb vesicles which encapsulate highly concentrated and

purified Hb solution (Hb purification; > 99.9 %, concentration; 36 g/dl) within a

phospholipid bilayer membrane as an artificial oxygen carrier 8-12. Ferrous Hb (Hb(Fe2+))

molecules in the Hb vesicles gradually lose their oxygen binding ability because they react

with ROS such as H2O2

13, 14 and nitrogen monoxide (NO*) 15 to become nonfunctional

ferric Hb, in addition to the autoxidation of oxyHb itself. The concentration of metHb in

the human erythrocyte is normally kept at less than 1 % by reduction systems such as

NADH-cytochrome b5, NADPH-flavin, and reductants such as glutathione and ascorbic

acid. Furthermore, ROS are eliminated by SOD, catalase, or peroxidase to suppress metHb

formation 16, 17. In contrast, metHb molecules generated in the Hb vesicle are not reduced

to the ferrous state due to the absence of the reduction systems, because the constitutive

enzymes and reductants necessary for metHb reduction in the human erythrocyte are

removed during Hb purification process 18. Furthermore, ROS elimination enzymes are

also completely removed in the process. For these reasons, the resulting Hb vesicles are

not effective against ROS, in particular, H2O2 which permeates the phospholipid bilayer

membrane of the Hb vesicles. We are in the process of constructing an H2O2 elimination

system using Hb vesicles in order to prolong the oxygen carrying ability of the vesicles.

Coencapsulation of catalase in the Hb vesicle is an effective method; however, it has some

practical problems such as the stability of catalase itself, the source and amount of catalase,

and loss of the preferred long-term (two years) preservation at room temperature. The

preservation period of donated blood is three weeks at 4 oC; therefore, the long-term

preservation of Hb vesicles is a desirable for pharmaceutical and medical reasons. Because

of these reasons, catalase is not used in Hb vesicles. Recently, we succeeded in the

suppression of the conversion of oxyHb to metHb in the Hb vesicle by coencapsulation of

metHb and L-Tyr in vesicles ((metHb/L-Tyr) Hb vesicles) and showed that this was

effective in vitro and in vivo 19. In the Hb vesicle, during the reaction of metHb with H2O2,

metHb itself is oxidized to ferrylHb radical. FerrylHb radical returns to the ferric state

(metHb) by electron donation from two L-Tyr molecules. This reaction is regarded as the

peroxidase activity of metHb. In the practical application of the method to the Hb vesicle,

we incorporated an H2O2 elimination system in the Hb vesicle. This is a unique idea in the

sense that “metHb is utilized for the suppression of metHb formation in the Hb vesicle”.

Chapter 4

- 66 -

Moreover, L-Tyr is a stable amino acid and is possible to encapsulate in the Hb vesicle.

We consider the (metHb/L-Tyr) Hb vesicles as “the second generation of Hb vesicles”.

However, the reduction process of ferrylHb radical to metHb by L-Tyr and its radical

behavior during the reduction are still unknown. In addition, the chemical properties of the

ferrylHb radicals are not well known. In this study, we analyzed the radical behavior of

ferrylHb radical and its reduction process by electron spin resonance (ESR) and UV-vis

spectrometry.

2. Reaction of metHb with H2O2 and generation of ferrylHb radical

2-1. Experimental section

MetHb was prepared by the reaction of HbCO (10 g/dl) with 2.5-fold excess

potassium ferricyanide in the heme base. The unreacted potassium ferricyanide and

ferrocyanide were removed by gel permeation chromatography (GPC, Sephadex-G25)

developed by PBS (pH7.4). Deferoxamine mesylate (1.6 mM) and L-Tyr (0 or 1 mM)

were added to the oxyHb or metHb solutions and these solutions were used as Hb samples.

Hb samples ([heme] = 20 μM) were reacted with 200 μM H2O2 ([heme]/[H2O2] = 1/10

molar ratio) in PBS (pH7.4 at 37 oC) and the reaction was monitored by the repetitive

scanning of a visible region from 300 to 700 nm at 2 min interval by using an UV-vis

spectrometer.

For the measurement of remained H2O2 concentrations, samples were reacted with

H2O2 under the same conditions and were periodically sampled out and the concentration

of H2O2

was determined spectrofluorometrically by measuring the amount of

6,6’-dihydroxy-[1,1’-biophenyl]-3,3’-diacetic acid (DBDA, Ex: 317 nm, Em: 405 nm),

generated by the horseradish peroxidase (HRP)-catalyzed reaction of

p-hydroxyphenylacetic acid (HPA) with H2O2. The final concentrations of HRP and HPA

were 4 μM and 6 mM, respectively, and the DBDA concentration was calculated after

separating the Hb samples by centrifugal filtration (cutoff 5 kDa , Ultrafree-MC, Millipore,

Bedford).

Chapter 4

- 67 -

2-2. Elimination of H2O2 by the metHb

We added H2O2 to the metHb solution (5 μM) with or without 1 mM L-Tyr. Figure

4-1 shows the changes of the H2O2 concentration in the metHb solutions with and without

L-Tyr. The initial elimination rates of H2O2 in the metHb solutions were calculated on the

basis of a pseudo first-order rate law. The elimination rates with and without L-Tyr at 37

oC were 3.0 x 103 and 3.2 x 104 M-1s-1, respectively. The apparent rate constant of the

metHb solution containing L-Tyr was about 10 times larger than that with the metHb

solution alone, and this value was estimated to be approximately equal to the 150 unit of

catalase. Moreover, around 40 μM H2O2 remained after a 30 min reaction of 200 μM

H2O2 in the metHb solution alone, whereas H2O2 was completely eliminated within 15

min in the metHb solution containing L-Tyr.

2-3. UV-vis measurement

The UV-vis spectral changes during the reaction of H2O2 in the metHb solution (5

μM) and the metHb solution containing L-Tyr (1 mM) are shown in Figure 4-2. In the case

of metHb alone, there was a shift in the spectrum of metHb, namely, the Soret band of

metHb (405 nm) was shifted to 417 nm, and the typical metHb peak at 630 nm was

completely abolished, which indicted a change to a different form of metHb. There was a

time-dependent decrease in peak intensities, indicating the degradation of this intermediate

form of Hb during the reaction of H2O2.

5

10

15

20

25

30

40

80

120

160

200

0

Time (min)

H

2

O

2

(μ

M)

5

10

15

20

25

30

40

80

120

160

200

0

Time (min)

H

2

O

2

(μ

M)

Figure 4-1 Time course of H2O2 elimination by (○) metHb (5 μM) (●) metHb

(5μM) + L-Tyr (1 mM) during the reaction with 200 μM of H2O2 at 37 oC.

Chapter 4

- 68 -

We speculated that this intermediate form of Hb was ferrylHb radical. It has been

reported that the ferrylHb radical is formed by the reaction of metHb with H2O2.

Furthermore, the stoichiometry of metHb (5 μM) and the reacted H2O2 (200 μM) suggests

that the metHb worked as enzyme such as catalase, although the stability of the enzyme

was low and it was degraded. Because the Fenton reaction can be prevented by the

addition of DFO, which is a chelator of Fe3+, this enzymatic H2O2 elimination was

considered to be a pseudo-catalase like reaction of metHb. Conversely, the metHb solution

with L-Tyr showed a fast spectral change and then stayed constant during the reaction as

shown in Figure 4-2(B). Also, the small spectral shift of the metHb solution with L-Tyr

suggested that the metHb reacted with H2O2 was converted to the ferrylHb radical. The

ratio of metHb to ferrylHb should be constant during reaction with H2O2. Comparing with

the reaction mechanism of peroxidase, where the ferric state is converted to the ferrylHb

radical state by the reaction of H2O2 to produce H2O, and then the ferryl radical state is

recovered to the ferric state by one electron oxidation of two substrate molecules to

generate another molecule of H2O, we could compare, in this case, the peroxidase and the

substrate to metHb and L-Tyr, respectively. More precisely, we could describe the

enzymatic reaction as reverse peroxidation, and the substrate as H2O2 where L-Tyr works

as an electron donor.

300

Wavelength (nm)

A

b

s (-)

700

500

300

700

500

0

1

2

3

0

1

2

3

(A)

(B)

300

Wavelength (nm)

A

b

s (-)

700

500

300

700

500

0

1

2

3

0

1

2

3

(A)

(B)

Figure 4-2 UV-vis spectral changes of 5 μM of metHb solutions ([heme] = 20 μM)

during the reaction with 200 μM H2O2 ([heme]:[H2O2] = 1/10) at 37 oC (a) metHb (b)

metHb + 1 mM L-Tyr. The scanning was performed at 4 min intervals, immediately

after the addition of H2O2 solution to each Hb solution.

Chapter 4

- 69 -

3. Preparation of ferrylHb radical and its radical mechanisms

3-1. Experimental section

FerrylHb radical was prepared by the reaction of metHb with H2O2. H2O2 (4.5

mM) was added to the metHb (420 μM) solution and stirred for 1 min at 25 oC. After the

reaction, the mixture was immediately cooled to 4 oC and the remaining H2O2 was

removed by GPC (Sephadex-G25) at 4 oC. MetHb and ferrylHb were identified with a

UV-vis spectrometry (V-570, Jasco, Tokyo, Japan) at 4 oC and an ESR apparatus (9.059

GHz, 3.00 mW, JES-TE200, JEOL Ltd., Tokyo, Japan) at 4 K and 25 K controlled by a

thermocontroller (MODEL 9650, Scientific Instrument Inc., Tokyo, Japan). The

specification of the ESR tube was 5 mm φ and 270 mm L (JEOL Ltd.).

3-2. UV-vis measurement

Figure 4-3 shows the UV-vis spectra of the prepared metHb and the isolated

ferrylHb after the reaction of metHb with H2O2 and the subsequent removal of H2O2. The

λmax of metHb in the Soret band at 405 nm shifted to 417 nm attributed to the λmax of

ferrylHb. Moreover, the inset of Figure 4-3 was the magnification of the Q band of the

ferrylHb, showing disappearance of the peak at 630 nm, which was the specific absorption

of metHb. These results indicated that metHb was completely converted to ferrylHb state

of Hb by reaction with H2O2. For the detection of ferrylHb by UV-vis spectrometry, the

temperature of the sample was strictly kept at 4 oC.

Figure 4-3 UV-vis spectra of 2 μM metHb and 2 μM ferrylHb after

isolation by GPC Inset is the migration of Q band region.

300

0.2

Time (min)

Ab

s (-)

0.4

0.6

0.8

0

1.0

400

500

600

700

metHb

ferrylHb

metHb

ferrylHb

450 500

600

700

0

0.05

0.1

300

0.2

Time (min)

Ab

s (-)

0.4

0.6

0.8

0

1.0

400

500

600

700

metHb

ferrylHb

metHb

ferrylHb

450 500

600

700

0

0.05

0.1

Chapter 4

- 70 -

3-3. ESR measurement

The ESR results are shown in Figure 4-4. The signal of the isolated ferrylHb

showed only a signal at g=6 attributed the high spin state (S = 5/2) of ferric heme under the

4 K condition with liquid He; however, we could not confirm the signal at g=2.006

attributed to the radical in the ferrylHb.

On the other hand, both signals at g=6 and g=2.006 were detected at 25 K. ESR results at

25 K showed the metHb signal at g=6 attributed to the ferric iron of the heme (Figure 4-5).

In the case of the isolated ferrylHb, we confirmed the appearance of the signal at g=2.006,

which was attributed to the signal of the ferryl radical of Hb, and the decrease of the signal

intensity at g=6. From the results of UV-vis. and ESR measurements, the isolated ferrylHb

possessed a radical in the molecule, therefore, it could be regarded as “ferrylHb radical”

generally shown as Hb(Fe4+=O*). Because the method produced to ferrylHb radical, we

defined and represented the radical state of Hb produced by the preparation and isolation

methods as ferrylHb radical from now on. We also confirmed that the ferrylHb radical

before the separation of H2O2 showed almost the same signal as after removal of H2O2,

suggesting that the ferrylHb radical can be isolated without any change of the radical state.

B (mT)

100

200

300

400

g = 2.006

g = 6

500

0

x 5

x 1

4 K

25 K

B (mT)

100

200

300

400

g = 2.006

g = 6

500

0

x 5

x 1

4 K

25 K

Figure 4-4 ESR spectra of isolated ferrylHb in PBS in the difference of

measurement temperature, 4 K and 25 K.

Chapter 4

- 71 -

3-4. Mechanisms of generation, isolation and disappearance of ferrylHb radical

Though the isolation and detection of ferrylHb radical was difficult, the generation

due to the reaction of heme protein with H2O2 is a well known phenomenon, and many

researchers have investigated the behaviors and characteristics of the radical using

recombinant Mb 20. FerrylHb radical and ferrylMb radical are known to be unstable

intermediates of Hb and Mb known as Compound I. Ferryl radical spontaneously converts

to the ferric state, known as autoreduction, and two tyrosines (Y103 and Y151) within the

ferrylMb radical participate in the autoreduction 21. In other studies, the globin radicals in

Hb or Mb generated by radical transfer from heme to the peptide chains were analyzed in

detail using recombinant Mb 22, 23. In particular, the two tyrosine residues (Y103 and

Y151) and/or a tryptophan residue (W14) of Mb were shown to be involved in the

generation of the globin radical 24-26. Although such theoretical studies regarding the

autoreduction mechanism have been reported, the application to utilize the ferrylHb

radical itself (e.g., enzymatic assay method, enzymatic synthesis) has not been reported.

Figure 4-5 ESR spectra of isolated and H2O2 remained ferrylHb radical in

PBS at 25 K.

B (mT)

100

200

300

400

isolated

ferrylHb radical

H2O2 remained

ferrylHb radical

metHb

g = 2.006

g = 6

500

0

x 5

x 1

x 5

g = 1.99

B (mT)

100

200

300

400

isolated

ferrylHb radical

H2O2 remained

ferrylHb radical

metHb

g = 2.006

g = 6

500

0

x 5

x 1

x 5

g = 1.99

Chapter 4

- 72 -

This because the ferryl radical is unstable for handling and the high reactivity comes from

the highly oxidative state of Compound I of heme-containing proteins.

Alayash et al. reported the isolation of ferrylMb (Mb(Fe4+=O). They reported the

kinetics of the ferrylMb generation using recombinant Mb 27. However, they reported only

the isolation of oxoferryl Mb, not the ferrylMb radical. We used their method of isolation

of ferrylMb to isolate the ferrylHb radical. We could completely convert metHb to

ferrylHb radical by reaction with H2O2 for 1 min at 25 oC. As shown in Figure 4-3, 4-4 and

4-5, we succeeded in the isolation of ferrylHb radical by cooling the solution down to 4 oC

after reaction at 25 oC and the removal of H2O2 with Sephadex G-25 by keeping the

temperature at 4 oC. The appropriate amount of H2O2 for the reaction with metHb was

determined to be a 2.5-fold molar excess relative to the heme of the metHb. This was

because the ferrylHb radical conversion did not proceed to completion by adding a 1:1

molar equivalent of H2O2 to heme, in spite of the prolongation of the reaction time to 10

min. Furthermore, Hb was denaturated in a 10-fold molar excess of H2O2. In addition, it

was technically impossible to remove such a large amount of H2O2 completely and rapidly

from the reaction mixture with GPC. From these considerations, we finally applied a

2.5-fold molar excess of H2O2 to the heme to prepare the ferrylHb radical. ESR

measurements at 25 K (Figure 4-4 and 4-5) confirmed the disappearance of the signal at

g=6 attributed to the high spin state (S = 5/2) of the ferric heme and the appearance of a

new signal at g=2.006 attributed to the ferrylHb radical, after the reaction of metHb with

H2O2. The ESR spectrum of the isolated ferrylHb radical was almost the same as that of

ferrylHb radical before the removal of H2O2, indicating that the H2O2 removal process

with GPC at 4 oC did not alter the molecule. In the ESR measurements shown in Figure

1B, it is interesting that the isolated ferrylHb radical showed only a signal at g=6 attributed

to ferric heme at 4 K; the signal at g=2.006 could not be detected. We could detect both

signals at g=6 and g=2.006 when the temperature was raised to 25 K by liquid He.

4. Temperature stability of ferrylHb radical

4-1. Experimental section

Temperature stability of the ferrylHb radical (2 μM) in PBS (pH7.4) was assessed

with UV-visible spectrometry by repetitive scanning in the visible region from 300 nm to

Chapter 4

- 73 -

700 nm at 2-minute intervals. The measurement was performed under strictly controlled

temperature conditions at 4 oC and 37 oC. The percentages of the changed ferrylHb radical

(the conversion of metHb) were calculated from the absorbance at 405 nm (λmax of

metHb).

4-2. Conversion of ferrylHb radical

The conversion of the ferrylHb radical to metHb was dependent on the temperature

(Figure 4-6). At 4 oC, the metHb conversion increased proportionally with time and finally

reached 25% after 60 min. Conversely, at 37 oC the conversion proceeded rapidly to 25%

within 6 min, and finally reached a maximum of 70 %. Therefore, the ferrylHb radical is

more amenable to manipulation at 4 oC.

4-3. Autoreduction of ferrylHb radical

The absorbance at 405 nm (λmax of metHb in the Soret band) of the isolated

ferrylHb radical was regarded as the baseline absorption of metHb (0%), and the

absorbance at 405 nm of the same concentration of pure metHb was regarded as 100%

metHb. The percentage of the absorbance increase from baseline was plotted with time at

4 oC and 37 oC. Hence we regarded this increase as the conversion of the ferrylHb radical

to metHb, namely, the autoreduction of the ferrylHb radical. The autoreduction

mechanism of ferrylMb was already reported 20, 21, 28. In the autoreduction process of

sperm whale ferrylMb, it is thought that Y103 and Y151 residues are involved in the

0

Time (min)

Con

version to

me

tHb

(%

)

10

20

30

40

50

60

20

40

60

80

37 oC

4 oC

0

Time (min)

Con

version to

me

tHb

(%

)

10

20

30

40

50

60

20

40

60

80

0

Time (min)

Con

version to

me

tHb

(%

)

10

20

30

40

50

60

20

40

60

80

37 oC

4 oC

Figure 4-6 Conversion of ferrylHb radical to metHb at 4 oC and 37 oC in PBS.

Chapter 4

- 74 -

reduction of the ferryl radical by two electrons donation. Interestingly, Y146 does not

contribute to the autoreduction even though it is closer to the heme iron than either Y151

or Y103. The autoreduction mechanism of the ferrylHb radical in this case is thought to be

similar to the mechanism of ferrylMb which involves intramolecular electron transfer to

heme from the globin chain amino acid components such as tyrosine residues (Y103 and

Y151) and a tryptophan residue (W14) 24-26. The rate of autoreduction was influenced by

the temperature. Autoreduction was slower at 4 oC than at 37 oC. Aggregation was

confirmed in the reaction mixture after UV-vis measurement at 37 oC. Such instability of

ferrylHb or its radical is well known to be related to both autoreduction and protein

denaturation accompanying the release of heme or Fe ion 7, 13. The isolated ferrylHb

radical itself was stable under low temperature conditions (1 – 4 oC); therefore, we could

stably handle it without significant autoreduction or denaturation by keeping low

temperatures. We hypothesized that the ferrylHb radical could be utilized as a stable H2O2

elimination system by a catalytic cycle.

5. Reduction of ferrylHb radical by L-Tyr

5-1. Experimental section

Reduction of the ferrylHb radical (2 μM) by an excess amount of L-Tyr (500 μM)

was assessed with the UV-vis spectrometry by repetitive scanning in the visible region

from 300 nm to 700 nm at 2-minute intervals at 4 oC. The percentage of the reduced

ferrylHb radical (the conversion of metHb) was calculated from the absorbance at 405 nm

(λmax of metHb). The reduction process of the ferrylHb radical was analyzed by ESR

method. Immediately after preparation of a ferrylHb radical solution (110 μM), it was

mixed with a solution of L-Tyr (1.8 mM) and stirred at 4 oC. ESR samples were taken

from the reaction mixture at 0, 1, 3, and 5 min., and each sample was immediately injected

into the ESR tube (5 mm φ, 270 mm L, JEOL Ltd.) and frozen at 196 K (liquid nitrogen).

5-2. Reduction of ferrylHb radical by L-Tyr (UV-vis measurement)

The conversion of the ferrylHb radical to metHb in the presence of L-Tyr at 4 oC is

shown in Figure 4-7. The ferrylHb radical was more rapidly converted to metHb in the

Chapter 4

- 75 -

presence of L-Tyr compared with the control (the ferrylHb radical solution without L-Tyr).

The conversion gradually increased and finally reached 65% after 60 min, whereas the

conversion in the control solution was 25 % after 60 min.

5-3. Reduction of ferrylHb radical by L-Tyr (ESR measurement)

Figure 4-8 shows the ESR signals during these reactions (in the presence and

absence of L-Tyr) at 0, 1, 3, and 5 minute time points. The signal of the control solution at

g=2.006, which was attributed to the ferryl radical of Hb, did not change in spite of the

reaction time, whereas the rapid radical disappearance and the metHb conversion were

confirmed in the L-Tyr-containing ferrylHb radical solution. The signal at g=6 gradually

increased, followed by the disappearance of the signal at g=2.006.

0

Time (min)

C

o

n

ve

rsion to

me

tH

b

(%

)

10

20

30

40

50

60

20

40

60

80

L-Tyr

control

0

Time (min)

C

o

n

ve

rsion to

me

tH

b

(%

)

10

20

30

40

50

60

20

40

60

80

L-Tyr

control

Figure 4-7 Conversion of ferrylHb radical to metHb by L-Tyr at 4 oC in PBS.

Figure 4-8 Time course changes of ESR spectra of 113 μM ferrylHb radical mixed with PBS

(control) and 900 μM L-Tyr.

g = 2.006

g = 6

B (mT)

100

200

300

400

500

0

B (mT)

100

200

300

400

500

0

g = 2.006

g = 6

0 min

0 min

1 min

1 min

3 min

3 min

5 min

5 min

A

B

g = 2.006

g = 6

B (mT)

100

200

300

400

500

0

B (mT)

100

200

300

400

500

0

B (mT)

100

200

300

400

500

0

B (mT)

100

200

300

400

500

0

B (mT)

100

200

300

400

500

0

B (mT)

100

200

300

400

500

0

g = 2.006

g = 6

0 min

0 min

1 min

1 min

3 min

3 min

5 min

5 min

A

B

Chapter 4

- 76 -

5-4. Radical mechanism of ferrylHb radical

We confirmed that the conversion of ferrylHb radical to metHb was enhanced in

the presence of L-Tyr. We hypothesized that the ferrylHb radical could accept two

electrons from two L-Tyr molecules, and that these L-Tyr molecules could form a bond at

their ortho-positions to produce dimerized L-Tyr (DiTyr). In considering the application of

this system to the Hb vesicle in vivo, the temperature of interest is 37 oC; however, the

experiments of reduction of ferrylHb radical by L-Tyr were performed at 4 oC in order to

prevent autoreduction of ferrylHb radical itself. It should be kept in mind that the

reduction of the ferrylHb radical by L-Tyr occurs more rapidly at 37oC than at 4 oC. The

mechanism of autoreduction is very complex, and is known to involve intra- and

intermolecular transfer of globin radicals. Lardinois et al. reported that the cross-linked

Mb dimmers were produced by coupling of the two Y151 tyrosine residues of two sperm

whale Mb molecules as a result of electron transfer from the Y151 residue to the ferryl

radical 29. In our study, the redox mechanisms involving the amino acid residues taking

part in the autoreduction of the ferrylHb radical are still unclear. However, the direct

reduction of highly oxidized heme by exogenous L-Tyr was shown to occur as evidenced

by the generation of diTyr as shown in Figure 4-9 which described in the next section.

Conversely, the autoreduced Hb molecules in our experiments might obey the rule of

intra- and intermolecular transfers of globin radical in spite of L-Tyr presence. It is

possible that the added L-Tyr was only involved in the direct reaction. Since metHb

converted to ferrylHb radical by the process of H2O2 elimination and the generated

ferrylHb radical was directly reduced to metHb by added L-Tyr, it was defined as a

catalytic reaction, and L-Tyr could be defined as a substrate in this catalytic reaction.

In the case of the ferrylHb radical alone (Figure 4-8(A)), the ESR signals after 1 –

5 minutes were almost the same as that of the sample at zero time. This indicates the

stability of the ferrylHb radial at 4 oC and agrees with the UV-vis measurement at 4 oC as

shown in Figure 4-6. This result also supports the notion that the autoreduction of the

ferrylHb radical was dependent on the temperature. In contrast, in the case of the ferrylHb

radical in the presence of L-Tyr (Figure 4-8(B)), the signal at g=2.006 attributed to the

ferrylHb radical almost disappeared within 1 min after the addition of L-Tyr at 4 oC. After

5 min, although the signal at g=2.006 changed little compared with 1 min and 3 min, the

signal at g=6 attributed to the high spin state of ferric iron (metHb) increased. Since the

autoreduction can be considered to be negligible under this reaction condition at 4 oC, the

Chapter 4

- 77 -

result reflects the reduction process of the ferrylHb radical to metHb by L-Tyr.

6. Analysis of L-Tyr in the reaction of metHb with H2O2

6-1. Experimental section

During the reaction of the metHb ([heme] = 20 μM) with H2O2 (200 μM,

[heme]/[H2O2] = 1/10) in PBS (pH7.4, at 37 oC), the reaction mixture was periodically

sampled out, and 200 μL catalase (5000 unit/mL) was added to eliminate H2O2. L-Tyr was

analyzed with an HPLC (LCsolution system, Shimadzu Co., Kyoto, Japan) equipped with

an ODS column (TSK-GEL ODS-80Ts, 4.6 mm I.D., 250 mm L, Tosoh Co., Tokyo,

Japan) after being separated from the Hb samples by centrifugal filtration (cutoff 5 kDa,

Ultrafree-MC, Millipore Co., MA, USA). The separated solutions were injected into the

column and developed with a mixed solvent (PBS/acetonitrile = 4/1, v/v) containing 0.3

vol% trifluoroacetic acid at a flow rate of 0.5 mL/min. The detection of solutes was

performed at 240 nm.

6-2. HPLC analysis of L-Tyr

The conversion of L-Tyr to the L-Tyr dimer (diTyr) during the reduction of the

ferrylHb radical to metHb was studied using high pressure liquid chromatography (HPLC),

(Figure 4-9). The peak attributed to L-Tyr gradually decreased and simultaneously a new

peak appeared between the retention times of 4 and 5 min. The new peak was identified as

diTyr by the comparison with the elution time of diTyr synthesized by the method of

Malencik et al 1. This clearly showed that L-Tyr reacted with the ferrylHb radical to

produce the diTyr.

6-3. Mechanism of diTyr generation

From the detection of diTyr in the reaction mixture of metHb with H2O2, it was

proven that the direct reduction of ferrylHb radical was occurred and the electrons were

donated from added L-Tyr. As the result, L-Tyr molecules which donated electrons to

ferrylHb radical were coupled with the ortho-positions between the two L-Tyr molecules.

Chapter 4

- 78 -

The concept was shown in Figure 4-10. FerrylHb radical generated by the reaction of

metHb with H2O2 accepted two electrons from two L-Tyr molecules. Then, the radical was

generated in the O atom of L-Tyr molecule called thyl radical. The radical was stabilized at

the ortho-position of L-Tyr molecule by the conjugated structure of radical location. As the

result, two molecules of L-Tyr which had the radical at ortho-position were coupled to

produce L-Tyr dimer called diTyr.

3

Time (min)

In

tensity (a

.u

.)

4

5

6

7

2

4

6

8

diTyr

0 min

20

40

60

L-Tyr

0

3

Time (min)

In

tensity (a

.u

.)

4

5

6

7

2

4

6

8

diTyr

0 min

20

40

60

L-Tyr

0

Figure 4-9 Time courses of L-Tyr changes on the reaction of metHb

with H2O2 at 37 oC in the presence of L-Tyr by HPLC.

metHb(Fe3+) + H2O2

Hb(Fe4+=O*)

metHb(Fe3+)

metHb(Fe3+) + H2O2

Hb(Fe4+=O*)

metHb(Fe3+)

Figure 4-10 Mechanism of L-Tyr dimer (diTyr) generation by the reaction of metHb

with H2O2 in the presence of L-Tyr.

Chapter 4

- 79 -

References

1. Malencik, D. A., Sprouse, J. F., Swanson, C. A., and Anderson, S. R., Dityrosine:

preparation, isolation and analysis, Anal. Biochem. 242 (1996) 202-213.

2. Giulivi, C., Romero, J. F., and Cadenas, E., The interaction of Trolox C, a

water-soluble vitamin E analog, with ferrylmyoglobin: reduction of the oxoferryl

moiety, Arch. Biochem. Biophys. 299 (1992) 302–312.

3. Giulivi, C., and Cadenas, E., Inhibition of protein radical reactions of ferrylmyoglobin

by the water-soluble analog of vitamin E, Trolox C, Arch. Biochem. Biophys. 303

(1993) 152–158.

4. Giulivi, C., and Cadenas, E., The reaction of ascorbic acid with different heme iron

redox states of myoglobin. Antioxidant and prooxidant aspects, FEBS Lett. 332

(1993) 287–290.

5. Galaris, D., Cadenas, E., and Hochstein, P., Redox cycling of myoglobin and

ascorbate: a potential protective mechanism against oxidative reperfusion injury in

muscle, Arch. Biochem. Biophys. 273 (1989) 497–504.

6. Giulivi, C., and Davies, J. K., Mechanism of the formation and proteolytic release of

H2O2-induced dityrosine and tyrosine oxidation products in hemoglobin and red blood

cells, J. Biol. Chem. 276 (2001) 24129-24136.

7. Nagababu, E., Ramasamy, S., Rifkind, J. M., Jia, Y., and Alaysh, A. I., Site-specific

cross-linking of human and bovine hemoglobins differentially alters oxygen binding

and redox side reactions producing rhombic heme and heme degradation,

Biochemistry 41 (2002) 7407-7415.

8. Sakai, H., Cabrales, P., Tsai, A. G., Tsuchida, E., and Intaglietta, M., Oxygen release

from low and normal P50 Hb vesicles in transiently occluded arterioles of the hamster

window model, Am. J. Physiol. 288 (2005) H2897-H2903.

9. Cabrales, P., Sakai, H., Tsai, A. G., Takeoka, S., Tsuchida, E., and Intaglietta, M.,

Oxygen transport by low and normal oxygen affinity hemoglobin vesicles in extreme

hemodilution, Am. J. Physiol. 288 (2005) H1885-H1892.

10. Sakai, H., Masada, Y., Horinouchi, H., Ikeda, E., Sou, K., Takeoka, S., Suematsu, M.,

Takaori, M., Kobayashi, K., and Tsuchida, E., Physiological capacity of the

reticuloendothelial system for the degradation of hemoglobin vesicles (artificial

oxygen carriers) after massive intravenous doses by daily repeated infusions for 14

days, J. Pharmacol. Exp. Ther. 311 (2004) 874-884.

Chapter 4

- 80 -

11. Yoshizu, A., Izumi, Y., Park, S. I., Sakai, H., Takeoka, S., Horinouchi, H., Ikeda, E.,

Tsuchida, E., and Kobayashi., K., Hemorrhagic shock resuscitation with an artificial

oxygen carrier, hemoglobin vesicle, maintains intestinal perfusion and suppresses the

increase in plasma tumor necrosis factor-α, ASAIO J. 50 (2004) 458-463.

12. Sakai, H., Takeoka, S., Park, S. I., Kose, T., Hamada, K., Izumi, Y., Yoshizu, A.,

Nishide, H., Kobayashi, K., and Tsuchida, E., Surface modification of hemoglobin

vesicles with poly(ethylene glycol) and effects on aggregation, viscosity, and blood

flow during 90 % exchange transfusion in anesthetized rats, Bioconjugate Chem. 8

(1997) 23-30.

13. Takeoka, S., Teramura, Y., Atoji, T., and Tsuchida, E., Effect of Hb-encapsulation

with vesicles on H2O2 reaction and lipid peroxidation, Bioconjugate Chem. 13 (2002)

1302-1308.

14. Teramura, Y., Kanazawa, H., Sakai, H., Takeoka, S., and Tsuchida E., Prolonged

oxygen-carrying ability of hemoglobin vesicles by coencapsulation of catalase in vivo,

Bioconjugate Chem. 14 (2003) 1171-1176.

15. Herold, S., and Rock, G., Reactions of deoxy-, oxy-, and methemoglobin with

nitrogen monoxide, J. Biol. Chem. 278 (2003) 6623-6634.

16. Shikama, K., A controversy on the mechanism of autoxidation of oxymyoglobin and

oxyhemoglobin: oxidation, dissociation, or displacement? Biochem. J. 223 (1984)

279-280.

17. Tomoda, A., Yoneyama, T., and Tsuji A., Changes in intermediate hemoglobins

during autoxidation of hemoglobin, Biochem. J. 195 (1981) 485-492.

18. Naito, Y., Fukutomi, I., Masada, Y., Sakai, H., Takeoka, S., Tsuchida, E., Abe, H.,

Hirayama, J., Ikebuchi, K., and Ikeda, H., Virus removal from hemoglobin solution

using Planova membrane, J. Artif. Organs. 5 (2002) 141-145.

19. Atoji, T., Aihara, M., Sakai, H., Tsuchida, E., and Takeoka S., Hemoglobin vesicles

containing methemoglobin and L-tyrosine to suppress methemoglobin formation in

vitro and in vivo, Bioconjugate Chem. 17 (2006) 1241-124.

20. Svistunenko, D. A., Dunne, J., Fryer, M., Nicholls, P., Reeder, B. J., Wilson, M. T.,

Bigotti, M. G., Cutruzzola, F., and Cooper, C. E., Comparative study of tyrosine

radicals in hemoglobin and myoglobins treated with hydrogen peroxide, Biophys. J.

83 (2002) 2845-2855.

Chapter 4

- 81 -

21. Lardinois, O. M., and Ortiz de Montellano, P. R., Autoreduction of ferryl myoglobin:

discrimination among the three tyrosine and two tryptophan residues as electron

donors, Biochemistry 43 (2002) 4601-4610.

22. Allentoff, A. J., Bolton, J. L., Wilks, A., Thompson, J. A., and Ortiz de Montellano, P.

R., Heterolytic versus hemolytic peroxide bond cleavage by sperm whale myoglobin

and myoglobin mutants, J. Am. Chem. Soc. 114 (1992) 9744-9749.

23. Wittig, P. K., Mauk, A. G., and Lay, P. A., Role of tyrosine-103 in myoglobin

peroxidase activity: kinetic and steady-state studies on the reaction of wild-type and

variant recombinant human myoglobins with H2O2, Biochemistry 41 (2002)

11495-11503.

24. Tew, D., and Ortiz de Montellano, P. R., Intramolecular translocation of the protein

radical formed in the reaction of recombinant sperm whale myoglobin with H2O2, J.

Biol. Chem. 263 (1988) 17880-17886.

25. Davies, M. J., Identification of a globin free radical in equine myoglobin treated with

peroxides, Biochem. Biophys. Acta. 1077 (1991) 86-90.

26. DeGray, J. A., Gunthur, M. R., Tschirrt-Gurh, R., Ortiz de Montellano, P. R., and

Mason, R. P., Peroxidation of a specific tryptophan of metmyoglobin by hydrogen

peroxide, J. Biol. Chem. 272 (1997) 2359-2362.

27. Alayash, A. I., Ryan, B. A. B., Eich, R. F., Olson, J. S., and Cachon, R. E., Reactions

of sperm whale myoglobin with hydrogen peroxide. Effect of distal pocket mutations

on the formation and stability of the ferryl intermediate, J. Biol. Chem. 274 (1999)

2029-2037.

28. Reeder, B. J., and Wilson, M. T., The effects of pH in the mechanism of hydrogen

peroxide and lipid hydroperoxide consumption by myoglobin: A role for the

protonated ferryl species, Free Rad. Biol. Med. 30 (2001) 1311-1318.

29. Lardinois, O. M., Ortiz de Montellano, P. R., Intra- and intermolecular transfers of

protein radicals in the reactions of sperm whale myoglobin with hydrogen peroxide, J.

Biol. Chem. 278 (2003) 36214-36226.

Chapter 4

- 82 -

Chapter 5

- 83 -

Chapter 5

Application of H2O2 Elimination System by MetHb and L-Tyr

to the Hb Vesicles

Contents

1. Introduction

2. Stepwise injection of H2O2 to metHb/L-Tyr added oxyHb solution

2-1. Experimental section

2-2. Effect of metHb/L-Tyr system

3. Autoxidation of metHb/L-Tyr added oxyHb solution

3-1. Experimental section

3-2. Effect of metHb/L-Tyr system

4. Preparation of the Hb vesicles containing metHb and L-Tyr

4-1. Experimental section

4-2. metHb/L-Tyr containing Hb vesicles ((metHb/L-Tyr) Hb vesicles)

5. Stepwise injection of H2O2 to a dispersion of (metHb/L-Tyr) Hb vesicles

5-1. Experimental section

5-2. Effect of metHb/L-Tyr coencapsulating to Hb vesicles

6. Autoxidation of (metHb/L-Tyr) Hb vesicles

6-1. Experimental section

6-2. Suppression of autoxidation

7. In vivo measurement of (metHb/L-Tyr) Hb vesicles

7-1. Experimental section

7-2. Effect in vivo

References

Chapter 5

- 84 -

1. Introduction

Hemoglobin (Hb)-based O2 carriers (HBOCs) as red blood cell substitutes have

been developed for clinical application in recent years 1-3. Their safety and usefulness have

been demonstrated by many researchers in various fields such as physiology, toxicology,

and biochemistry 4-6. The demand for HBOCs has been increasing year by year due to

limitations of donated blood, such as infectious agents, shortage and storage issues.

HBOCs are generally classified into two types: one is the acellular-type which

comprises directly modified Hb molecules such as cross-linked Hb 7, polymerized Hb 8,

and polymer-conjugated Hb 9. Some of the acellular-type HBOCs have advanced to phase

III clinical trials 10, 11. Another kind is the cellular-type Hb systems such as Hb vesicles 12

or liposome-encapsulated Hb 13, in which Hb molecules are encapsulated with a

phospholipid bilayer membrane. Although they are not yet in clinical trials, they have been

shown to have excellent O2 carrying ability and good safety profiles in vivo

10, 14-18.

Ferrous Hb (Hb(Fe2+)) molecules of HBOCs gradually lose their O2 binding ability,

because they react with reactive oxygen species (ROS) such as hydrogen peroxide (H2O2)

19-20 or nitrogen monoxide (NO*) 21 to become nonfunctional ferric Hb (Hb(Fe3+), metHb),

in addition to the autoxidation of oxyHb molecules themselves. In humans, the

concentration of metHb molecules in the red blood cells is usually kept below 1 % by

reduction systems such as NADH-cytochrome b5, NADPH-flavin, glutathione, and

ascorbic acid. Furthermore, ROS are eliminated by several mechanisms such as

superoxide dismutase (SOD), catalase, and peroxidase that help to prevent the metHb

formation 23-24. In contrast, metHb generated in the Hb vesicle are not reduced to the

ferrous state due to the absence of the reduction systems, because constitutive enzymes

and reductants necessary for metHb reduction are removed during the Hb purification

process 25. The half-life of oxyHb of the Hb vesicles was 14 hr in vivo, and more than

90 % of oxyHb was converted to metHb within 48 hr in the case of 20 vol% top loading to

rats 20. In human plasma, the concentration of H2O2 is reported to be 4-5 μM 26 and to

elevate to as much as 100-600 μM under inflammatory 27 or ischemia-reperfusion

conditions 28. Therefore, Hb vesicles are required to have an H2O2 elimination system for

effective prolongation of their O2 carrying ability. We previously reported that H2O2

generated in the living body was the main reason of metHb formation of the Hb vesicles,

and the O2 carrying ability of the Hb vesicles in which catalase was coencapsulated was

vastly prolonged in vivo by elimination of H2O2

20. Regarding the catalase-coencapsulated

Chapter 5

- 85 -

vesicles, it is somewhat difficult to obtain a sufficient amount of human catalase from

human donated blood. Furthermore, the catalase activity is gradually lost due to

denaturation during storage at 25 oC.

Horseradish peroxidase, which also eliminates H2O2 in its enzymatic reaction, is

converted to a ferrylHb radical Hb(Fe4+=O*) state after reaction with H2O2, and the radical

state is returned to the ferric state by acceptance of two electrons from substrates such as

p-hydroxyphenylacetic acid (HPA) 29, 30. This is a well-known reaction in an H2O2 assay

method. L-Tyrosine (L-Tyr) dimer or polymer can also be synthesized by an

HRP-catalyzed oxidation 31, 32. We considered from the above mechanism that if metHb,

which has ferric heme like HRP does, could also function as an H2O2 elimination enzyme

in the presence of L-Tyr as a substrate. MetHb can easily be prepared from purified Hb for

the preparation of vesicles. Moreover, L-Tyr itself is amino acid with high stability and

good cost performance. In this Chapter, we prepared Hb vesicles coencapsulating metHb

and L-Tyr thereby incorporating the metHb elimination system into the Hb vesicles

themselves. We then evaluated the effect of this coencapsulation on the suppression of

metHb formation. Furthermore, we evaluated the safety of the vesicles in vivo.

2. Stepwise injection of H2O2 to metHb/L-Tyr added oxyHb solution

2-1. Experimental section

L-Tyr (0, 1 mM) and prepared metHb solution (0, 0.5 g/dl) were added to the

oxyHb solutions (5 g/dl, PBS (pH7.4)), and then they were incubated and stirred, and

H2O2 (340 μM) was continued to add to the solutions at 10 min intervals. Just before the

each addition, their samples were sampled out and immediately added 20 μL of catalase

solution (5000 unit/mL) to dismutate remaining H2O2. Percentage of metHb was

periodically measured using a standard cyanomethemoglobin method. The same

experiment was done using Hb solution (5.5 g/dl, PBS (pH7.4)) containing 10 % metHb

formed by autoxidation.

Chapter 5

- 86 -

2-2. Effect of metHb/L-Tyr system

Figure 5-1 shows the time course of metHb formation of oxyHb or metHb/L-Tyr

added oxyHb solution by stepwise injection of H2O2 (310 μM) in 10-min intervals.

H2O2 (310 μM) injection to oxyHb (control solution, [oxyheme]=3.1 mM) showed

an elevation in the metHb percentage with increasing injections of H2O2, and the

percentage of metHb reached 95 % after 60 min (6 injections). Whereas the metHb and

L-Tyr added oxyHb (Figure 5-1 (●), metHb: L-Tyr = 0.5 g/dl: 1 mM) showed a significant

suppression of metHb formation in the vesicles. The metHb percentage of the solution was

40 % after 60 min (6 injections). On the other hand, only metHb added oxyHb solution (■)

did not showed the suppression effect of metHb formation. Furthermore, only L-Tyr added

oxyHb solution (□) also did not showed the suppression effect. Because the mechanism

was based on the reduction of generated ferrylHb radical by the added L-Tyr, the

peroxidase cycle which continued to eliminate H2O2 was not worked in the absence of

metHb or L-Tyr

0

Time (min)

me

tH

b (%

)

10

20

30

40

50

60

20

40

60

80

100

10

H2O2 injection

(310 μM)

0

Time (min)

me

tH

b (%

)

10

20

30

40

50

60

20

40

60

80

100

10

H2O2 injection

(310 μM)

Figure 5-1 Time course of metHb formation of oxyHb at 37 oC during the

stepwise addition of H2O2 (310 μM) to 5 g/dl oxyHb solution containing (●) 0.5

g/dl metHb and 1 mM L-Tyr, (■) 0.5 g/dl metHb, (□) 1 mM L-Tyr, (○) no

metHb and L-Tyr (control).

Chapter 5

- 87 -

3. Autoxidation of metHb/L-Tyr added oxyHb solution

3-1. Experimental section

To the oxyHb solutions (5 g/dl, PBS (pH7.4)) containing pyridoxal 5’-phosphate

(PLP, 1.9 mM, Merck Co.) as an allosteric effector of oxygen affinity of Hb were added

L-Tyr (0, 1 mM) and prepared metHb solution (0, 0.25, 0.5 g/dl), and then they were

incubated and shaken (120 times/min) under the oxygen partial pressure (pO2) of 40 Torr

at 37 oC. The Percentage of metHb was periodically measured by standard

cyanomethemoglobin method.

3-2. Effect of metHb/L-Tyr system

Figure 5-2 shows the autoxidation of oxyHb solutions added metHb and L-Tyr

under the pO2 of 40 Torr at 37 oC. When the pO2 is approximately the P50 of Hb, the rate

of metHb formation tends to show a maximum, and the average oxygen partial pressure in

mixed venous blood is estimated to be 40 Torr. Therefore, we used the constant oxygen

partial pressure of 40 Torr to measure the metHb formation by autoxidation of the oxyHb

solution of the oxyHb vesicle dispersion for estimation of the in vivo behavior.

0

Time (min)

me

tH

b

(%

)

6

12

18

24

30

36

20

40

60

80

100

10

0

Time (min)

me

tH

b

(%

)

6

12

18

24

30

36

20

40

60

80

100

10

Figure 5-2 Time course of autoxidation of oxyHb solution (5 g/dl) under pO2 of

40 Torr at 37 oC. (●) 0.5 g/dl metHb and 1 mM L-Tyr, (■) 0.25 g/dl metHb and

1 mM L-Tyr, (○) no metHb and L-Tyr (control).

Chapter 5

- 88 -

On the control sample (oxyHb only), the percentage of metHb was gradually

increased following time passing, T50 (the time that the half of oxyHb was converted to

metHb) was about 18 hr. On the other hand, oxyHb solution added metHb and L-Tyr

showed the dramatic suppression of metHb formation by autoxidation, T50 of the sample

solutions added metHb (0.25 g/dl (■) and 0.5 g/dl (●)) were 33 hr and 38 hr, respectively.

It would be considered that the H2O2 generated by the dismutation of O2

-* produced by the

autoxidation was eliminated by the metHb/L-Tyr system. As a result, it was suggested that

metHb/L-Tyr suppressed the “secondary” metHb formation by the H2O2 generated by the

dismutation of O2

-* produced by the autoxidation of oxyHb. The acceleration of metHb

formation was confirmed on the oxyHb solutions added metHb and L-Tyr (●, ■) after 18hr

from measurement start. It would be considered that added L-Tyr was consumed, and the

activity of the peroxidase cycle depended on the concentrations of metHb and L-Tyr would

be weak.

4. Preparation of the Hb vesicles containing metHb and L-Tyr

4-1. Experimental section

MetHb was prepared by the reaction of HbCO (10 g/dl) with an excess amount of

potassium ferricyanide. The unreacted potassium ferricyanide and ferrocyanide were

removed by ultrafiltration (cutoff Mw 50 kDa, ADVANTEC, Tokyo) until the

concentration of potassium ferricyanide was less than 1 μM by monitoring the absorbance

with the UV-vis spectrometer. The metHb solution was concentrated to 40 g/dl using a 50

kDa cutoff filter as noted above. HbCO solutions containing 5 or 10 mol% metHb were

prepared by mixing the concentrated metHb solution with a 40 g/dl HbCO solution.

Pyridoxal 5’-phosphate (PLP, Sigma, St. Louis, MO) was added to the Hb solution as an

allosteric effector at a 2.5 equimolar ratio of PLP to HbCO. The L-Tyr solution prepared

previously was added to 0.2 N NaOH and the resulting solution were added to the

HbCO/metHb solution. The final concentrations of Hb and L-Tyr in the HbCO/metHb

solution were adjusted to 40 g/dl and 1.0 or 8.5 mM, respectively. To prepare the Hb

vesicles, powders of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylchorine (DPPC),

cholesterol, 1,5-bis-O-hexadecyl-N-succinyl-L-glutaminate (DHSG) (Nippon Fine

Chemical Co.), and 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-PEG5000

Chapter 5

- 89 -

(PEG-DSPE, NOF Co., Tokyo) were mixed at a molar ratio of 5:5:1:0.033 and added to

the HbCO/metHb solution and the mixture was stirred at 25 oC for 12 hr. The resulting

dispersion of the multilamellar vesicles was subsequently extruded through the

nitrocellulose membrane filters with a pore size of 0.22 μm (Fuji Film Co., Tokyo) to

prepare the Hb vesicles with an average diameter of 261 ± 30 nm. After the separation of

unencapsulated Hb by ultracentrifugation (10000g, 60 min), the precipitate of the Hb

vesicles was redispersed into saline in order to adjust the Hb concentration of the Hb

vesicle dispersion to 10 g/dl. HbCO within the vesicles was decarbonyzed and oxygenated

to HbO2 by irradiation of visible light onto a liquid film under O2 atmosphere.

4-2. metHb/L-Tyr containing Hb vesicles ((metHb/L-Tyr) Hb vesicles)

The solubility of L-Tyr in pure water is 1.2 mM; however, we succeeded in

preparation of an Hb solution containing 8.5 mM L-Tyr by dissolving L-Tyr to an alkali

solution (NaOH) for the pH adjuster of Hb solution. It might be contributed the interaction

of L-Tyr with highly concentrated Hb molecule (40 g/dl). As a result, we succeeded in the

construction of Hb vesicles having an H2O2 elimination system using Hb only as protein

or enzyme. The concept is shown in Figure 5-3.

coencapsulation

H

2

O

2

Hb(Fe3+)

Hb(Fe4+=O*)

2L-Tyr

Tyr-Tyr,

H2O

H

2

O

Hb vesicle

peroxidase activity of metHb with L-Tyr

enzymatic cycle

coencapsulation

H

2

O

2

Hb(Fe3+)

Hb(Fe4+=O*)

2L-Tyr

Tyr-Tyr,

H2O

H

2

O

Hb vesicle

peroxidase activity of metHb with L-Tyr

enzymatic cycle

Figure 5-3 Concept of utilizing peroxidase activity (H2O2 elimination enzymatic cycle)

of metHb by L-Tyr to Hb vesicle.

Chapter 5

- 90 -

5. Stepwise injection of H2O2 to a dispersion of (metHb/L-Tyr) Hb vesicles

5-1. Experimental section

A dispersion of (metHb/L-Tyr) Hb vesicles in PBS (pH7.4) coencapsulating metHb

and L-Tyr (metHb : L-Tyr = 2 g/dl : 1 mM, 4 g/dl : 1 mM, or 4 g/dl : 8.5 mM) was

incubated with stirring, and H2O2 (310 μM, Ultra Pure Grade, Kanto Chemical Co. Tokyo)

was injected in stepwise injection into the solutions at 10 min intervals. Just before each

injection, 20 μl of the dispersion of (metHb/L-Tyr) Hb vesicles was sampled out and 20 μl

of a catalase solution (50000 unit) was immediately added for the elimination of the

remaining H2O2. The percentage of metHb in the (metHb/L-Tyr) Hb vesicles was

periodically calculated by the ratio of absorbance at 405 nm (metHb) and 430 nm

(deoxyHb) in the Soret band using a UV-vis spectrometer without destruction of the Hb

vesicles.

5-2. Effect of metHb/L-Tyr coencapsulating to Hb vesicles

Figure 5-4 shows the time course of metHb formation in the conventional Hb

vesicles or the (metHb/L-Tyr) Hb vesicles by stepwise injection of H2O2 (310 μM) in

10-min intervals.

0

10

Time (min)

me

tH

b

(in

H

bV

)

(%

)

20

40

60

420

20

40

60

80

100

50

0

10

Time (min)

me

tH

b

(in

H

bV

)

(%

)

20

40

60

420

20

40

60

80

100

50

Figure 5-4 Time course of metHb formation in Hb vesicles during the stepwise

addition of H2O2 (310 μM) to 5 g/dl Hb vesicle dispersion coencapsulating ○ no

metHb and L-Tyr, ● 4 g/dl metHb and 1 mM L-Tyr, □ 2 g/dl metHb and 1 mM

L-Tyr, ■ 4 g/dl metHb and 8.5 mM L-Tyr at 37 oC.

Chapter 5

- 91 -

H2O2 (310 μM) injection to a 5 g/dl conventional Hb vesicle dispersion

([heme]=3.1 mM) showed an elevation in the metHb percentage with increasing injections

of H2O2, and the percentage of metHb reached 85 % after 60 min (6 injections). Whereas

the (metHb/L-Tyr) Hb vesicles (metHb: L-Tyr = 2 g/dl: 1 mM, 4 g/dl: 1 mM) showed a

significant suppression of metHb formation in the vesicles. The metHb percentages of

these Hb vesicles were 67 % and 50 % after 60 min (6 injections), respectively.

Furthermore, by increasing the amount of L-Tyr to 8.5 mM (metHb: 4 g/dl), metHb

formation was dramatically suppressed; the percentages were 17 % and 45 %, after 60 min

and 420 min, respectively (6 and 42 injections, respectively).

From these results, it was confirmed that the formation of metHb from oxyHb by

reaction with H2O2 was suppressed by the H2O2 elimination system of metHb and L-Tyr.

Moreover, the persistence of the metHb suppression effect depends on the amount of

metHb as an enzyme and L-Tyr as a substrate.

6. Autoxidation of (metHb/L-Tyr) Hb vesicles

6-1. Experimental section

A dispersion of (metHb/L-Tyr) Hb vesicles in PBS (pH7.4) coencapsulating metHb

and L-Tyr (metHb : L-Tyr = 2 g/dl : 1 mM, 4 g/dl : 1 mM, or 4 g/dl : 8.5 mM)was

incubated and shaken (120 times/min) under pO2 of 40 Torr at 37 oC. The percentage of

metHb in the vesicles was periodically measured with a UV-vis spectrometry.

6-2. Suppression of autoxidation

Besides the formation of metHb by H2O2, oxyHb is automatically oxidized to

metHb, initiated by one-electron reduction of coordinated dioxygen molecule to generate

O2

-*. MetHb formation in vesicles by such autoxidation was measured under the condition

of the pO2 of 40 Torr at 37 oC, because at this pO2 value the rate of metHb formation is

maximal. In the conventional Hb vesicles, the percentage of metHb periodically increased

by autoxidation of oxyHb in the vesicles. The T50 was 13 hr and the metHb percentage

reached 90 % after 24 hr incubation (Figure 5-5). On the other hand, the metHb formation

Chapter 5

- 92 -

of the Hb vesicles containing 4 g/dl metHb and 1 mM L-Tyr was effectively suppressed;

the T50 was 24 hr. Furthermore, in the Hb vesicles containing 4 g/dl metHb and 8.5 mM

L-Tyr, the percentage of metHb formed was 20 % and 43 % after 24 hr and 48 hr,

respectively, and did not reach 50 % when the measurement ended (48 hr).

Autoxidation of oxyHb is also an important factor of metHb formation. The

autoxidation rate of oxyHb depends on many factors such as the O2 affinity; P50, the pO2,

temperature and pH. OxyHb molecules in the Hb vesicles are also influenced by these

same factors, though the rates could be different from that of the oxyHb solution. The

half-life of oxyHb to metHb (T50) in the conventional Hb vesicles (P50 = 33 Torr) at 37 oC

was about 12 hr, shorter than that of the oxyHb solution (T50 = 24 hr, PBS, pH7.4). The

autoxidation rate of the Hb vesicles containing metHb and L-Tyr was also effectively

reduced in a similar way to that observed in the above experiment of H2O2 stepwise

addition. The reason could be that the autoxidation of oxyHb is additionally influenced by

H2O2. The O2

-* generated by the oxyHb autoxidation is spontaneously converted to H2O2

in the presence of H+. This H2O2 attacks the other oxyHb molecules. Therefore, the

elimination of the H2O2 would result in the suppression of the autoxidation rate of oxyHb

in the Hb vesicles.

0

50

Time (hr)

me

tH

b

(in

H

bV

)

(%

)

12

24

36

48

20

40

60

80

100

0

50

Time (hr)

me

tH

b

(in

H

bV

)

(%

)

12

24

36

48

20

40

60

80

100

Figure 5-5 Time course of metHb formation in Hb vesicles during autoxidation of 5

g/dl Hb vesicle dispersion coencapsulating ○ no metHb and L-Tyr, ● 4 g/dl

metHb and 1 mM L-Tyr, □ 2 g/dl metHb and 1 mM L-Tyr and ■ 4 g/dl metHb

and 8.5 mM L-Tyr under pO2 of 40 Torr at 37 oC.

Chapter 5

- 93 -

7. In vivo measurement of (metHb/L-Tyr) Hb vesicles

7-1. Experimental section

Wistar rats (body weight: 240 - 260 g) were anesthetized with diethyl ether, and a

preparation of (metHb/L-Tyr) Hb vesicles containing 4 g/dl of metHb and 8.5 mM of

L-Tyr was injected into the tail vein (20 ml/kg, n = 6). Blood was withdrawn from the tail

vein and centrifuged (12000g, 5 min) to collect the vesicle fraction in the supernatant. The

percentage of metHb within the vesicles was measured with a UV-vis spectrometry. The

number of blood cells was measured with a blood cell counter (Sysmex, KX-21, Kobe).

7-2. Effect in vivo

Figure 5-6 shows the percentage of generated metHb after the intravenous injection

(20 ml/kg) of the conventional Hb vesicles or the (metHb/L-Tyr) Hb vesicles into the tail

vein of Wistar rats (n = 6). The metHb percentages of the conventional Hb vesicles and the

(metHb/L-Tyr) Hb vesicles containing metHb/L-Tyr after 4 hr were 22 % and 18 %,

respectively, and the T50 were 14 hr and 44 hr, respectively. Furthermore, the Wistar rats

were all alive for 72 hr after the measurement at which time they were sacrificed,

indicating the safety of this system in vivo. These results confirmed the suppressive effect

of metHb formation in vivo using the (metHb/L-Tyr) Hb vesicles.

24

0

Time (hr)

me

tH

b

(in

H

b

V

)

(%

)

36

48

60

12

72

20

40

60

80

100

50

24

0

Time (hr)

me

tH

b

(in

H

b

V

)

(%

)

36

48

60

12

72

20

40

60

80

100

50

Figure 5-6 Time course of metHb formation in Hb vesicles in vivo (20 ml/kg,

Wistar rat). Hb vesicles were coencapsulating ○no metHb and L-Tyr, ●4 g/dl

metHb and 8.5 mM L-Tyr.

Chapter 5

- 94 -

In a previous study, we coencapsulated catalase to eliminate H2O2 generated in

vivo thus suppressing the formation of metHb within Hb vesicles, and we confirmed the

suppression of metHb formation 20 (Figure 5-7). The T50 of the catalase-coencapsulated Hb

vesicles was 37 hrs with a catalase concentration of 4.2 x 104 unit/ml (0.5 g/dl catalase as a

protein concentration). We estimated that the concentration of catalase was the amount at

the saturation point of T50 prolongation, because the same T50 was obtained when 5.6 x 104

unit/ml catalase was used for coencapsulation. Compared with these results, the

(metHb/L-Tyr) system was more effective in the suppression of metHb formation by

elimination of H2O2. It is thought that catalase activity is gradually lost at 37 oC in vivo

20,

whereas the activity of the (metHb/L-Tyr) system should be very stable at 37 oC, and the

amount of L-Tyr (8.5 mM within the Hb vesicle) was sufficient to eliminate H2O2 in the

Hb vesicles. From these results, the increase of the metHb percentage of the

(metHb/L-Tyr) Hb vesicles is likely due to the autoxidation of Hb which is impossible to

suppress in this system.

0

20

40

60

80

100

0

5 10 15 20 25 30 35 40

Time (hr)

metHb

(%

)

50

HbV 1

HbV 6

HbV 4

HbV 3

HbV 2

HbV 5

HbV 7

0

20

40

60

80

100

0

5 10 15 20 25 30 35 40

Time (hr)

metHb

(%

)

50

HbV 1

HbV 6

HbV 4

HbV 3

HbV 2

HbV 5

HbV 7

Figure 5-7 Changes of metHb percentages in the catalase-coencapsulating Hb

vesicles in vivo. The concentrations of coencapsulating catalase were 1, 0 unit/ml

(control), 2, 2.8x103 unit/ml, 3, 8.4x103 unit/ml, 4, 1.7x104 unit/ml, 5, 2.8x104

unit/ml, 6, 4.2x104 unit/ml, and 7, 5.6x104 unit/ml.

Chapter 5

- 95 -

References

1. Tsuchida E., Artificial Red Cells, John Willy and Sons, New York (1995).

2. Winslow R. M., Progress on blood substitutes, Nature Med. 3 (1997) 474-474.

3. Chang, T. M. S., Blood Substitutes: Principles, Methods, Products and Clinical Trials,

S. Karger AG, Switzerland (1997).

4. Tsuchida E. Ed., Blood Substitutes: present and future perspectives, Elsevier Science,

Amsterdam (1998).

5. Reiss, J. G., Oxygen carriers (“blood substitutes”)-Raison d'Etre, Chemistry, and

Some Physiology, Chem. Rev. 101 (2001) 2797-2920.

6. D’Agnillo F. and Alayash A. I., Redox cycling of diaspirin cross-liked hemoglobin

induced G2/M arrest and apoptosis in cultured endothelial cells, Blood 98 (2001)

3315-3323.

7. Przybelski, R. J., Dairy, E. K., and Birnbaum, M. L., The pressor of effect of

hemoglobin – good or bad?, In Advances in Blood Substitutes: Industrial

Opportunities and Medical Challenges (Winslow, R. M., Vandegriff, K. D., and

Intaglietta, M., Eds.) Birkhauser, Boston (1997).

8. Gould, S. A., Moore, E. E., Hoyt, D. B., Burch, J. M., Haenel, J. B., Garcia, J.,

DeWoskin, R., Moss, G. S., The first randomized trial of human polymerized

hemoglobin as a blood substitute in acute trauma and emergency surgery, J. Am. Coll.

Surg. 187 (1998) 113-122.

9. McCarthy, M. R., Vandegriff, K. D., and Winslow, R. M., The role of facilitated

diffusion in oxygen transport by cell-free hemoglobins: implications for the design of

hemoglobin-based oxygen carriers, Biophys. Chem. 92 (2001) 103-117.

10. Mullon, J., Giacoppe, G., Clagett, C., McCune, D., and Dillard, T., Transfusions of

polymerized bovine hemoglobin in a patient with severe sutoimmune hemolytic

anemia, N. Engl. J. Med. 342 (2000) 1638-1643.

11. Sloan, E. P., Koenigsberg, M., Gens, D., Cipolle, M., Runge, J., Mallory, M. N., and

Rodman, G. Jr., Diaspirin cross-linked hemoglobin (DCLHb) in the treatment of

severe traumatic hemorrhagic shock: a randomized controlled efficacy, JAMA 282

(1999) 1857-1864

Chapter 5

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12. Sakai, H., Horinouchi, H., Tomiyama, K., Ikeda, E., Takeoka, S., Kobayashi, K., and

Tsuchida, E., Hemoglobin-vesicles as oxygen carriers, Influence on phagocytic

activity and histopathological changes in reticuloendothelial system, Am. J. Pathol.

159 (2001) 1079-1088.

13. Rudolph, A. S., Sulpizio, A., Hieble, P., MacDonald, V., Chavez, M., and Feuerstein,

G., Liposome encapsulating attenuates hemoglobin-induced vasoconstriction inrabbit

arterial segment, J. Appl. Physiol. 82 (1997) 1826-1835.

14. Sakai, H., Cabrales, P., Tsai, A. G., Tsuchida, E., and Intaglietta, M., Oxygen release

from low and normal P50 Hb vesicles in transiently occluded arterioles of the hamster

window model, Am. J. Physiol. 288 (2005) H2897-H2903.

15. Cabrales, P., Sakai, H., Tsai, A. G., Takeoka, S., Tsuchida, E., and Intaglietta, M.,

Oxygen transport by low and normal oxygen affinity hemoglobin vesicles in extreme

hemodilution, Am. J. Physiol. 288 (2005) H1885-H1892.

16. Sakai, H., Masada, Y., Horinouchi, H., Ikeda, E., Sou, K., Takeoka, S., Suematsu, M.,

Takaori, M., Kobayashi, K., and Tsuchida, E., Physiological capacity of the

reticuloendothelial system for the degradation of hemoglobin vesicles (artificial

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days, J. Pharmacol. Exp. Ther. 311 (2004) 874-884.

17. Yoshizu, A., Izumi, Y., Park, S., Sakai, H., Takeoka, S., Horinouchi, H., Ikeda, E.,

Tsuchida, E., and Kobayashi, K., Hemorrhagic shock resuscitation with an artificial

oxygen carrier, hemoglobin vesicle, maintains intestinal perfusion and suppresses the

increase in plasma tumor necrosis factor-α, ASAIO J. 50 (2004) 458-463.

18. Sakai, H., Takeoka, S., Park, S. I., Kose, T., Hamada, K., Izumi, Y., Yoshizu, A.,

Nishide, H., Kobayashi, K., and Tsuchida, E., Surface modification of hemoglobin

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flow during 90% exchange transfusion in anesthetized rats, Bioconjugate Chem. 8

(1997) 23-30.

19. Takeoka, S., Teramura, Y., Atoji, T., and Tsuchida, E., Effect of Hb-encapsulation

with vesicles on H2O2 reaction and lipid peroxidation, Bioconjugate Chem. 13 (2002)

1302-1308.

Chapter 5

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20. Teramura, Y., Kanazawa, H., Sakai, H., Takeoka, S., and Tsuchida, E., Prolonged

oxygen-carrying ability of hemoglobin vesicles by coencapsulation of catalase in vivo,

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monoxide, J. Biol. Chem. 278 (2003) 6623-6634.

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hemoglobin: A venerable puzzle, Chem. Rev. 98 (1998) 1357-1373.

23. Shikama, K. A controversy on the mechanism of autoxidation of oxymyoglobin and

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279-280.

24. Tomoda A., Yoneyama, T., and Tsuji, A., Changes in intermediate hemoglobins

during autoxidation of hemoglobin, Biochem. J. 195 (1981) 485-492.

25. Naito, Y., fukutomi, I., Masada, Y., Sakai, H., Takeoka, S., Tsuchida, E., Abe, H.,

Hirayama, J., Ikebuchi, K., and Ikeda, H., Virus removal from hemoglobin solution

using Planova membrane, J. Artif. Organs. 5 (2002) 141-145.

26. Nagababu, E., and Rifkind, J. M., Reaction of Hydrogen Peroxide with

Ferrylhemoglobin: Superoxide Production and Heme Degradation, Biochemistry 39

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27. Gunther, M. R., Sampath, V., and Caughey, W. S., Potential roles of myoglobin

autoxidation in myocardial ischemia-reperfusion injury, Free Radic. Biol. Med. 26

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28. Svistunenko, D. A., Patel, R. P., Voloshchenko, S. V., and Wilson, M. T., The

globin-based free radical of ferryl hemoglobin is detected in normal human blood, J.

Biol. Chem. 272 (1997) 7114-7121.

29. Hofmann, F., Hofmann, L., Hubl, W., Meissner, D., Determination of horseradish

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the fluorescence enzyme immunoassay, Z. Med. Lab. Diagn. 25 (1984) 14-21.

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hydrogen peroxide: involvement of ferryl intermediate, Biochemistry 26 (1987)

6684-6688.

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31. Fukuoka, T., Tachibana, Y., Tonami, H., Uyama, H., and Kobayashi, S., Enzymatic

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Preparation, isolation, and analysis, Anal. Biochem. 242 (1996) 202-213.

Chapter 6

- 99 -

Chapter 6

Extension of H2O2 Elimination System by metHb

for the Various Applications

Contents

1. Introduction

2. FerrylHb radical reduction to metHb by various substrates

2-1. Experimental section

2-2. Conversion to metHb from ferrylHb radical

3. H2O2 elimination by the metHb/substrate system

3-1. Experimental section

3-2. Effect of metHb/substrate system

References

Chapter 6

- 100 -

1. Introduction

In chapter 2-6, we described the various things for the application to Hb vesicles.

These experiments, analyses and discussion in the chapters were performed on the

assumption that they were applied to Hb vesicles. The problem of metHb formation in Hb

vesicles was settled by the success of metHb/L-Tyr Hb vesicles in vivo though the safety

and the preparation method had to be more studied 1-3.

Here, we tried to extend the H2O2 elimination system by metHb for the various

applications to not only Hb vesicles but also the others. However, only Hb was the heme

proteins which we could reasonably obtain on a massive scale. Therefore, it would be

thought that use of the other heme proteins was not practical. From the reasons, we

decided on metHb as heme protein for use for the extension of H2O2 elimination system.

Therefore, substrate was required to change for the purpose. In this chapter, we described

the effects of substrates for the peroxidase activity of ferrylHb radical genetrated by the

reaction of metHb with H2O2.

2. FerrylHb radical reduction to metHb by various substrates

2-1. Experimental section

FerrylHb radical was prepared by the reaction of metHb with H2O2. H2O2 (4.5

mM) was added to the metHb (420 μM) solution and stirred for 1 min at 25 oC. After the

reaction, the mixture was immediately cooled to 4 oC and the remaining H2O2 was

removed by GPC (Sephadex-G25) at 4 oC. The reduction processes of the ferrylHb radical

by carious substrates were analyzed by ESR method. Immediately after preparation of a

ferrylHb radical solution (110 μM), it was mixed with a solution of a substrate (1.8 mM)

and stirred for 30 sec at 4 oC. The reaction was performed by using various substrates.

Each sample was immediately injected into the ESR tube (5 mm φ, 270 mm L, JEOL

Ltd.) and frozen at 196 K (liquid nitrogen). The percentages of conversion to metHb from

ferrylHb radical were calculated from the integration values of peak areas of metHb and

ferrylHb radical on ESR measurements. The structures of substrates are shown in Figure

6-1.

Chapter 6

- 101 -

2-2. Conversion to metHb from ferrylHb radical by substrates

The percentages of conversions to metHb from ferrylHb radical by various

substrates are shown in Table 6-1. The percentages of the conversions were the average of

several times measurement.

First, the difference between L- and D- of Tyr was not confirmed. From the results

of phenylalanine (Phe) and Tyr, it was considered that the 4-hydroxyl group of phenyl ring

played an important rule of the electron donation to ferrylHb radical. If it was not exist, the

reduction should be occurred. Furthermore, it was confirmed the w-time value of

conversion percentage was confirmed when the substrate was 3,4-dihydroxyphenylalanine

(L-DOPA) compared with L-Tyr. L-DOPA has two hydroxyl groups at 3 and 4 positions of

Figure 6-1 Structures of substrates used for the ferrylHb radical reduction to

metHb by the peroxidase activity of ferrylHb radical.

NH2

OH

O

HO

L-tyrosine

NH2

NH

O

HO

L-tryptophan

O

HO

OH

p-hydroxyphenylpropionic acid

O

HO

OH

p-hydroxyphenylacetic acid

NH2

OH

O

HO

OH

L-DOPA

NH2

OH

O

HO

NH2

OH

O

HO

OCH3

NO2

methoxy-Tyr

nitro-Tyr

NH2

O

HO

L-phenylalanine

N

H

O

HO

3-indole-acetic acid

NH

O

HO

3-indole-propionic acid

NH2

SH

O

OH

L-cysteine

HS

NH

NH2

HO

O

O

NH

HO

O

O

glutathione

Chapter 6

- 102 -

phenyl ring of L-Phe 4. L-Tyr has one hydroxyl group at 4 position. From the

consideration,

it was assumed that the resonance structures were formed more easily in the L-DOLA than

L-Tyr because of the election richness 5. As a result, the electron donations to ferrylHb

radical were occurred more smoothly when the substrate was L-DOPA than L-Tyr.

We assumed the larger conversion of ferrylHb radical to metHb by methoxy-L-Tyr

compared with L-Tyr, which had methoxy group as an electron acceptation part. However,

the result was reverse, the smaller conversion to metHb from ferrylHb radical was

confirmed. It would be considered that the three dimensional obstruction of bulky

methoxy group suffocated the approach of hydroxyl group of phenyl ring to heme pocket.

In reverse, we assumed the smaller conversion of ferrylHb radical to metHb by nitro-L-Tyr

compared with L-Tyr, which had nitro group as an electron donation part. Surely, the

conversion was smaller than L-Tyr.

The remarkable ferrylHb radical conversion to metHb was confirmed in the

experiments using compounds including indole group as substrates. The detail mechanism

was not will known, indole compounds were suitable to the substrates for the ferrylHb

radical reduction to metHb. From these results, the possible candidates were appeared.

Table 6-1 percentages of conversions to metHb

from ferrylHb radical by the various substrates.

conversion to

metHb / %

L-Tyr

19.9

D-Tyr

18.8

p-HPP

32.1

p-HPA

22.8

L-Phe

0.8

D-Phe

0.0

L-DOPA

53.2

D-DOPA

41.3

nitro-L-Tyr

17.3

methoxy-L-Tyr

12.9

L-Trp

29.8

D-Trp

27.9

L-Cys

0.7

3-IPA

3-IAA

substrate

36.8

52.6

conversion to

metHb / %

L-Tyr

L-Tyr

19.9

D-Tyr

D-Tyr

18.8

p-HPP

p-HPP

32.1

p-HPA

p-HPA

22.8

L-Phe

L-Phe

0.8

D-Phe

D-Phe

0.0

L-DOPA

L-DOPA

53.2

D-DOPA

D-DOPA

41.3

nitro-L-Tyr

17.3

methoxy-L-Tyr

12.9

L-Trp

L-Trp

29.8

D-Trp

D-Trp

27.9

L-Cys

L-Cys

0.7

3-IPA

3-IAA

substrate

36.8

52.6

Chapter 6

- 103 -

Indole substrates (3-indole-propionic acid (IPA) and 3-indole-acetic acid (IAA)) showed

the larger conversion compared with L- and D-Trp. It would be considered that the

difference was due to the three-dimensional obstruction by amino group of L- and D-Trp 6,

7. From this reason, 3-IPA and 3-IAA would approach to heme pocket compared with L-

and D-Trp.

Interestingly, the ferrylHb radical was not converted to metHb by L-Cys as a

substrate. It would be considered that the direct reduction of ferrylHb radical by reductant

(L-Cys) was not occurred. If the experimental phenomenon was under anaerobic condition,

L-Cys reduced metHb to ferrousHb 8, 9. However, ferrylHb radical which was higher

oxidative state than metHb was not reduced to ferrylHb, metHb or ferrousHb. The reason

was not unknown, it was very interesting result.

3. H2O2 elimination by the metHb/substrate system

3-1. Experimental section

Hb samples ([heme] = 20 μM) were reacted with 200 μM H2O2 ([heme]/[H2O2] =

1/10 molar ratio) in PBS (pH7.4 at 37 oC). For the measurement of remained H2O2

concentrations, samples were reacted with H2O2 under the same conditions and were

periodically sampled out and the concentration of H2O2 was determined by measuring the

amount of 6,6’-dihydroxy-[1,1’-biophenyl]-3,3’-diacetic acid (DBDA, Ex: 317 nm, Em:

405 nm), generated by the horseradish peroxidase (HRP)-catalyzed reaction of

p-hydroxyphenylacetic acid (HPA) with H2O2. The final concentrations of HRP and HPA

were 4 μM and 6 mM, respectively, and the DBDA concentration was calculated after

separating the Hb samples by centrifugal filtration (cutoff 5 kDa, Ultrafree-MC,

Millipore).

3-2. Effect of metHb/substrate system

Figure 6-2 shows the changes of the H2O2 concentration in the metHb solutions in

the presence of various substrates. It was confirmed that 40 μM H2O2 remained after a 30

min reaction of 200 μM H2O2 in the metHb solution alone, whereas H2O2 was completely

eliminated within 30 min in the metHb solution in the presence of substrates. It was

indicated that the added substrate were worked as the electron donors to ferrylHb radical in

Chapter 6

- 104 -

the peroxidase cycles of ferrylHb. The H2O2 elimination rates by various substrates tended

to agree with the conversion percentages calculated from ESR results shown in Table 6-1,

however, the perfect relationships between the conversion percentages and H2O2

elimination rate were not confirmed. It would be considered that the generated radical

species of substrates might suffocate the enzymatic cycle. In fact, the experiments of

oxyHb protection from H2O2 by metHb/substrate system showed the bad profiles in the

particular substrates data not shown).

50

100

150

200

0

5

10

15

20

25

30

Time / min

concentra

tion o

f H

2

O

2

/μ

M

control

L- Trp

L- Tyr

3- IAA

3- IPA

50

100

150

200

0

5

10

15

20

25

30

Time / min

concentra

tion o

f H

2

O

2

/μ

M

control

L- Trp

L- Tyr

3- IAA

3- IPA

Figure 6-2 Time course of H2O2 elimination by metHb in the presence of

various substrates during the reaction with 200 μM of H2O2 at 37 oC.

Chapter 6

- 105 -

References

1. Atoji, T., Aihara, M., Sakai, H., Tsuchida, E., and Takeoka S., Hemoglobin vesicles

containing methemoglobin and L-tyrosine to suppress methemoglobin formation in

vitro and in vivo, Bioconjugate Chem. 17 (2006) 1241-124.

2. Takeoka, S., Teramura, Y., Atoji, T., and Tsuchida, E., Effect of Hb-encapsulation

with vesicles on H2O2 reaction and lipid peroxidation, Bioconjugate Chem. 13 (2002)

1302-1308.

3. Teramura, Y., Kanazawa, H., Sakai, H., Takeoka, S., and Tsuchida E., Prolonged

oxygen-carrying ability of hemoglobin vesicles by coencapsulation of catalase in vivo,

Bioconjugate Chem. 14 (2003) 1171-1176.

4. Schallreuter, K. U., Wazir, U., Kothari, S., Gibbons, N. C., Moore, J., and Wood, J.

M., Human phenylalanine hydroxylase is activated by H2O2: a novel mechanism for

increasing the L-tyrosine supply for melanogenesis in melanocytes, Biochem. Biophys.

Res. Commun. 322 (2004) 88-92.

5. Metodiewa, D., and Dunford, H. B., The role of myeloperoxidase in the oxidation of

biologically active polyhydroxyphenols (substituted catechols), Eur. J. Biochem. 193

(1990) 445-448.

6. Petersen, F. N., Jensen, M. O., and Nielsen, C. H., Interfacial tryptophan residues: a

role for the cation-pi effect?, Biophys. J. 89 (2005) 3985-96.

7. Scheiner, S., Kar, T., and Pattanayak, J., Comparison of various types of hydrogen

bonds involving aromatic amino acids, J. Am. Chem. Soc. 124 (2002) 13257-64.

8. Takeoka, S., Ohgushi, T., Sakai, H., Kose, T., Nishide, H., and Tsuchida, E.,

Construction of artificial methemoglobin reduction systems in Hb vesicles, Artif.

Cells Blood Sub. Immobil. Biotechnol. 25 (1997) 31-41.

9. Takeoka, S., Sakai, H., Kose, T., Mano, Y., Seino, Y., Nishide, H., and Tsuchida, E.,

Methemoglobin formation in hemoglobin vesicles and reduction by encapsulated

thiols, Bioconjugate Chem. 8 (1997) 539-44.

Chapter 6

- 106 -

Chapter 7

- 107 -

Chapter 7

Conclusions and Future Prospects

Contents

1. Conclusions

2. Future prospects

Chapter 7

- 108 -

1. Conclusions

In this thesis, the mechanisms of the interaction of oxyHb and oxyHb vesicles with

H2O2 were clarified, and the supremacy of the cellular structure of Hb vesicles over

acellular type HBOCs was proven. However, oxyHb molecules in the Hb vesicles were

finally oxidized to metHb and lost oxygen carrying ability. Hb vesicles have been

developed for the oxygen carrier. Even if the safety and supremacy of Hb vesicles are

shown, the prolongation of oxygen carrying ability has to be realized for the clinical

application. For the purpose, I attempted the metHb reduction to ferrous Hb in the Hb

vesicles by using membrane permeability of reductants. However, it was inhabited by the

autoxidation of reductants themselves to form H2O2.

From these results, I understood the necessity of H2O2 elimination. Catalase was

not used by the reasons of the sources, stability and safety. From the various tries and

errors, I found the metHb/L-Tyr system for the H2O2 elimination system. It was the

reduction of ferrylHb radical to metHb by the peroxidase activity of Hb itself. I

investigated the characteristics of the ferrylHb radical generated by the reaction of metHb

with H2O2. And I established the isolation method of ferrylHb radical. FerrylHb radical

was autoreduced to metHb at 37 oC, the reduction would be involved in the intermolecular

L-Tyr residues or the other amino residues. On the other hand, the autoreduction was

widely suppressed at 4 oC. It was indicated that the radical was activated at higher

temperature. I confirmed the direct reduction of ferrylHb radical to metHb at 4 oC in the

presence of L-Tyr. It was also proven that diTyr was detected in the reaction mixture of

metHb with H2O2 in the presence of L-Tyr. As a result, I constructed the H2O2 elimination

system by metHb and L-Tyr, and the possibility of the application to Hb vesicle of this

mechanism was indicated.

I prepared Hb vesicles coencapsulating metHb and L-Tyr thereby incorporating the

metHb elimination system into the Hb vesicles themselves. I then evaluated the effect of

this coencapsulation on the suppression of metHb formation in vitro and in vivo. The

results showed very good profiles, the suppression of metHb formation in Hb vesicles was

confirmed by the elimination H2O2. From these results, I succeeded in the suppression

system of metHb formation in Hb vesicles using only Hb and L-Tyr.

The problem of metHb formation in Hb vesicles was settled by the success of

metHb/L-Tyr Hb vesicles.

Chapter 7

- 109 -

2. Future Prospects

In present, Hb vesicles have been developed for the clinical trial, and the safety

and efficacies have been proven by the many field’s researchers such as chemistry,

biology, medicine and etc. I think that Hb vesicles as a material are the sheer dint of

efforts. However, I must not satisfy the present condition. The demands for artificial

oxygen carriers from various fields changes following the time passing. Therefore, I have

to widely know the environment around our researches.

The suppression of metHb in Hb vesicles will more developed by the findings of

new substrate. For the success, the screening technique and methodology of analyses will

be required. Moreover, metHb reduction system in Hb vesicles may be realized by the

development of H2O2 elimination system.

If the safety for the clinical application can be ignore, the prolongation of oxygen

carrying ability by the chemical method may be easy. However, it is not able to ignore, it

is rather important than the prolongation of oxygen carrying ability. I think the point is the

most difficult thing in the biomaterial researches.

Finally, I believe that this thesis contributed, is contributing and will contribute to

the development of Hb vesicles in the past, present and future.

- 110 -

Academic Achievement

(研究報文)

1. Tomoyasu Atoji, Hirotaka Yatami, Motonari Aihara and Shinji Takeoka, “Effect of

L-tyrosine on the peroxidase activity of ferrylhemoglobin radical”, Free Rad. Biol.

Med., submitted.

2. Tomoyasu Atoji, Motonari Aihara, Hiromi Sakai, Eishun Tsuchida and Shinji

Takeoka, “Hemoglobin vesicles containing methemoglobin and L-tyrosine to suppress

methemoglobin formation in vitro and in vivo”, Bioconjugate Chem. 17 (2006)

1241-1245.

3. Shinji Takeoka, Yuji Teramura, Tomoyasu Atoji and Eishun Tsuchida, “Effect of

Hb-encapsulation with vesicles on H2O2

reaction and lipid peroxidation”,

Bioconjugate Chem. 13 (2002) 1302-1308.

(総説)

1. 阿閉友保, 岡村陽介, 武岡真司, “動く臓器としての血液に学ぶ －人工赤血

球・人工血小板への挑戦－”, ファイバー スーパーバイオミメティックス

(2006) NTS 出版.

(特許)

1. 武岡真司, 土田英俊, 酒井宏水, 寺村裕治, 阿閉友保, “メト化防止剤を含有す

る人工酸素運搬体”, 特願 2004-309268.

2. 土田英俊, 武岡真司, 寺村裕治, 阿閉友保, “酸素運搬体システム、人工酸素運

搬体、および還元剤”, 特願 2003-570874.

(国際学会)

1. Tomoyasu Atoji, Motonari Aihara, Hiromi Sakai, Shinji Takeoka and Eishun

Tsuchida, “Prolongation oxygen carrying ability of hemoglobin vesicles by hydrogen

peroxide elimination using methemoglobin and L-tyrosine”, 10th International

Symposium on Blood Substitutes, (RhodeIsland, June. 2005).

2. Tomoyasu Atoji, Yuji Teramura, Shinji Takeoka and Eishun Tsuchida. “Reduction of

methemoglobin within a vesicle by using membrane permeation of reductants”, 9th

International Symposium on Blood Substitutes, (Tokyo, May. 2003).

その他、連名で 5 件、計 7 件発表。

- 111 -

(国内学会)

1. 阿閉友保, 谷田海博孝, 相原源就, 武岡真司, 土田英俊, “L-チロシンによるフ

ェリルヘモグロビンラジカル還元反応の解析とヘモグロビン小胞体への応用”,

第 55 回高分子年次会, (名古屋, 2006 年 5 月).

2. 阿閉友保, 相原源就, 武岡真司, 土田英俊, “L-チロシンによるフェリルヘモグ

ロビン還元反応の解析(1) ”, 日本化学会第 86 回春季年会, (船橋, 2006 年 3 月).

3. 阿閉友保, 相原源就, 武岡真司, 土田英俊, “活性酸素消去系の導入によるヘモ

グロビン小胞体の酸素運搬能向上”, 第54回高分子討論会, (山形, 2005年9月).

4. 阿閉友保, 相原源就, 武岡真司, 土田英俊, “metHb/L-Tyr による過酸化水素消

去系を封入した Hb 小胞体の機能評価”, 第 12 回日本人工血液代替物学会年次

大会, (東京, 2005 年 6 月).

5. 阿閉友保, 相原源就, 武岡真司, 土田英俊, “メトヘモグロビン/L-チロシン内包

ヘモグロビン小胞体のメト化抑制効果”, 第 54 回高分子年次会, (横浜, 2005 年

5 月).

6. 阿閉友保, 武岡真司, 土田英俊, “過酸化水素消去系を有したヘモグロビン小胞

体の評価”, 第 53 回高分子討論会, (札幌, 2004 年 9 月).

7. 阿閉友保, 武岡真司, 土田英俊, “L-チロシン内包によるヘモグロビン小胞体の

メト化抑制” (ワークショップ), 第 11 回日本人工血液代替物学会年次大会, (札

幌, 2004 年 7 月).

8. 阿閉友保, 寺村裕治, 武岡真司, 西出宏之, 土田英俊, “カタラーゼ共封入ヘモ

グロビン小胞体の機能評価”, 日本化学会第83回春季年会, (東京, 2003年3月).

9. 阿閉友保, 寺村裕治, 武岡真司, 西出宏之, 土田英俊, “カタラーゼを内包した

Hb 小胞体の活性酸素に対する安定性” (パネルディスカッション), 第 40 回日

本人工臓器学会, (札幌, 2002 年 10 月).

10. 阿閉友保, 寺村裕治, 武岡真司, 西出宏之, 土田英俊, “還元剤の膜透過性を利

用したメトヘモグロビン小胞体の還元”, 第 81 回日本化学会春季年会, (東京,

2002 年 3 月).

その他、連名で 12 件、計 22 件発表。

- 112 -

- 113 -

Acknowledgement

The presented thesis is the collection of the studies which have been carried out under

the guidance of Prof. Dr. Shinji Takeoka, Department of Polymer Chemistry in Waseda

University, during 2001 – 2007. The author expresses the greatest acknowledgement to Prof.

Dr. Shinji Takeoka for his valuable suggestions, discussions and continuous encouragement

throughout this study. The author also expresses his sincere gratitude to Professor Dr.

Hiroyuki Nishide for his valuable advice and encouragement as well as kindest supports on

the work. The author thanks Professor Dr. Kiyotaka Sakai, as an expert of biomedical

engineering, Department of Chemical Engineering, for his effort as a member of the judging

committee for the doctoral thesis. Sincere gratitude is expressed to Professor Dr. Eishun

Tsuchida in Advanced Research Institute for Science and Engineering, Waseda University.

The author acknowledges to Dr. Yuji Teramura for his valuable and continuous

support in the experiments and discussion. The author also acknowledges to Assoc. Prof. Dr.

Hiromi Sakai and Assoc. Prof. Dr. Keitarou Sou for their pertinent advices and kind supports.

It is also acknowledged to Assoc. Prof. Dr. Teruyuki Komatsu, Lecturer Dr. Akito

Nakagawa and Dr. Yosuke Okamura, for their advice, remarks and discussion.

The author is very much indebted to active colleagues, Dr. H. Ohkawa, Messrs. S.

Arai, S. Ishihara, I. Takemura, Y. Obata, S. Ishihara, Y. Masada, Y. Naito, H. Kanazawa, S.

Hisamoto, K. Kubota, M. Aihara, M. IIzuka, D. Suzuki, T. fujie, Y. Mochizuki, S. Inenaga,

T. Gotoh, A. Satoh, Y. Furuki, H. yatami, and Misses. I. Sato, M. Moritake, N. Ohmochi, Y.

Oguro, S. Utsunomiya, N. Ikegaya, K. Kaiyama. And the author would like to thank deeply

all the members of the Laboratory who have helped him kindly.

Finally, the author expresses his gratitude heartily to his parents Mr. Yoshimitsu Atoji,

Mrs. Hiromi Atoji, his sister Mrs. Yuki Yamagiwa, his grandfather and mothers Mrs. Misao

Atoji, Mr. Takakazu Fujimoto, Mrs. Yayoi Fujimoto for their affectionate contributions.

January 2007

Tomoyasu Atoji