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Suppression of Methemoglobin Formation in Hemoglobin Vesicles Using Peroxidase Activity of Methemoglobin Tomoyasu ATOJI
Page 1
Suppression of Methemoglobin Formation in Hemoglobin Vesicles
Using Peroxidase Activity of Methemoglobin
A Thesis
Presented to
Waseda University
March 2007
Tomoyasu ATOJI

Page 2
Promoter:
Prof. Dr. Shinji Takeoka
Referees:
Prof. Dr. Hiroyuki Nishide
Prof. Dr. Kiyotaka Sakai

Page 3
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

Page 4

Page 5
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

Page 6
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

Page 7
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

Page 8
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

Page 9
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

Page 10
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

Page 11
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.

Page 12
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.

Page 13
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

Page 14
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.

Page 15
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

Page 16
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.

Page 17
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.

Page 18
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.

Page 19
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

Page 20
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.

Page 21
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).

Page 22
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- 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

Page 23
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.

Page 24
Chapter 1
- 16 -
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37. Caswell, J. E., Strange, M.B., Rimmer, D. M. 3rd, Gibson, M. F., Cole, P., and Lefer,
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38. Fitzpatrick, C. M., Savage, S. A., Kerby, J. D., Clouse, W. D., and Kashyap, V. S.,
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
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Page 31
Chapter 2
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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

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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

Page 33
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.

Page 34
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.

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- 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

Page 36
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- 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.

Page 37
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

Page 38
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.

Page 39
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.

Page 40
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

Page 41
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

Page 42
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
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
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.

Page 43
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.

Page 44
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.

Page 45
Chapter 2
- 37 -
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Page 49
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

Page 50
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.

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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.

Page 52
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 -) 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)

Page 53
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

Page 54
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

Page 55
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)

Page 56
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- 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.

Page 57
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- 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.

Page 58
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.

Page 59
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
-

Page 60
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- 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

Page 61
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- 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.

Page 62
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- 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
-

Page 63
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- 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.

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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.

Page 65
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.

Page 66
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.

Page 67
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

Page 68
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.

Page 69
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.

Page 70
Chapter 3
- 62 -

Page 71
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

Page 72
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

Page 73
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”.

Page 74
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).

Page 75
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
M)
5
10
15
20
25
30
40
80
120
160
200
0
Time (min)
H
2
O
2
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.

Page 76
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.

Page 77
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

Page 78
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.

Page 79
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

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- 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

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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.

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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
max 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

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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

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- 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

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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.

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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.

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- 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.

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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
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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.

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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.

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Chapter 5
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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

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Chapter 5
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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

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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.

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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).

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- 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).

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- 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

Page 97
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.

Page 98
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.

Page 99
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

Page 100
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.

Page 101
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- 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.

Page 102
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- 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.

Page 103
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- 95 -
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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
peroxidase (HRP) with the substrates H2O2 and p-hydroxyphenylacetic acid (HPA) in
the fluorescence enzyme immunoassay, Z. Med. Lab. Diagn. 25 (1984) 14-21.
30. Yusa, K., and Shikama, K., Oxidation of oxymyoglobin to metmyoglobin with
hydrogen peroxide: involvement of ferryl intermediate, Biochemistry 26 (1987)
6684-6688.

Page 106
Chapter 5
- 98 -
31. Fukuoka, T., Tachibana, Y., Tonami, H., Uyama, H., and Kobayashi, S., Enzymatic
polymerization of tyrosine derivatives. peroxidase- and protease-catalyzed synthesis
of poly(tyrosine)s with different structures, Biomacromolecules 3 (2002) 768-774.
32. Malencik, D. A., Sprouse, J. F., Swanson, C. A., and Anderson, S. R., Dityrosine:
Preparation, isolation, and analysis, Anal. Biochem. 242 (1996) 202-213.

Page 107
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

Page 108
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.

Page 109
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

Page 110
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

Page 111
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

Page 112
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
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
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.

Page 113
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.

Page 114
Chapter 6
- 106 -

Page 115
Chapter 7
- 107 -
Chapter 7
Conclusions and Future Prospects
Contents
1. Conclusions
2. Future prospects

Page 116
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.

Page 117
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.

Page 118
- 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。

Page 119
- 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。

Page 120
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Page 121
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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