Kinetics of CO and O2 Binding to Human Serum Albumin-Heme Hybrid

in the second and fifth binding sites of FeP in the albumin structure. The absorption ... the O2 photodissociation of rHSA-FeP(8) also showed the same...
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Bioconjugate Chem. 2000, 11, 772−776

Kinetics of CO and O2 Binding to Human Serum Albumin-Heme Hybrid Teruyuki Komatsu, Yasuko Matsukawa, and Eishun Tsuchida* Department of Polymer Chemistry, Advanced Research Institute for Science & Engineering, Waseda University, Tokyo 169-8555, Japan. Received February 14, 2000; Revised Manuscript Received April 21, 2000

The kinetics of the CO and O2 binding to the synthetic hemoprotein, recombinant human serum albumin (rHSA) incorporating eight 2-[8-{N-(2-methylimidazolyl)}octanoyloxymethyl]-5,10,15,20tetrakis(o-pivalamido)phenylporphinatoiron(II)s (FePs) [rHSA-FeP(8)] have been investigated by laser flash photolysis. Time dependence of the absorption change accompanied the CO rebinding to rHSAFeP(8) was composed of three phases. The fastest component was the axial base elimination, and the long-lived biphasic decay corresponds to the direct recombination of CO to the five-N-coordinated FePs in rHSA. The rate constants of the fast and slow phases of the CO association [kCO on (fast), 6 -1 s-1 and 6.7 × 105 M-1 s-1, respectively. The initial kCO on (slow)] were determined to be 4.9 × 10 M amplitude after the laser pulse gave the concentration ratio of the fast and slow phases (n ) 3); (i) two of the eight FePs exhibited the slow rate constants and (ii) they are presumably accommodated in the second and fifth binding sites of FeP in the albumin structure. The absorption decay following the O2 photodissociation of rHSA-FeP(8) also showed the same behavior. Thermodynamically, the large ∆Gq of the slow phase of the CO rebinding, which mainly comes from the enthalpic factor, suggests the appearance of additional steric hindrance on the central metal iron of FeP. Furthermore, orientation of the porphyrin plane in rHSA was predicted by molecular simulation, which supports the experimental data from the kinetic observations.

INTRODUCTION

The binding kinetics of small gaseous ligands (CO, O2, or NO) to hemoproteins, e.g., myoglobin (Mb) and hemoglobin (Hb), have been widely studied for many years and it is still of current interest for the development of their mutant derivatives (Gibson 1970; Olson et al., 1971, 1988; Sawicki et al., 1977; Sharma et al., 1976; Steinmeier et al., 1975). For instance, the main features of the binding processes of Mb can be described as a scheme including two intermediate states in which the ligand molecule is inside the protein, but has not yet bound to the iron center of the heme. Much research has been carried out to interpret these transient states in terms of the stereochemistry of the Mb molecule (Ansari et al., 1985; Austin et al., 1975; Jongeward et al., 1988; Sato et al., 1990). A variety of theoretical simulations have also been undertaken to study the response of the ligand and protein structure to the photodissoiation (Case et al., 1979; Kottalam et al., 1988). We have recently found that the recombinant human serum albumin (rHSA) incorporating the tetraphenylporphinatoiron(II) derivative linked a covalently bound axial base, 2-[8-{N-(2-methylimidazolyl)}octanoyloxymethyl]-5,10,15,20-tetrakis(R,R,R,R-o-pivalamido)phenylporphinatoiron(II) (FeP) (rHSA-FeP) can reversibly bind and release O2 under physiological conditions (in aqueous media, pH 7.3, 37 °C) like Hb and Mb (Komatsu et al., 1999; Tsuchida et al., 1997, 1999). It is noteworthy that the obtained rHSA-FeP hybrid shows a good compatibility with the human whole blood and can quantitatively transport O2 in vivo (Tsuchida et al., 2000). * To whom the correspondence should be addressed. CREST investigator, JST. Phone: +81 3-5286-3120. Fax: +81 3-32054740. E-mail: [email protected].

That is, rHSA-FeP is now one of the most promising materials as a totally synthetic red cell substitute. Interestingly, laser flash photolysis to the dioxygenated rHSA-FeP leads to nonexponential relaxation of the O2photodissociated products on a microsecond time scale (Tsuchida et al., 1999). This paper describes the rebinding kinetics of CO and O2 to this synthetic hemoprotein and its thermodynamic properties. Furthermore, molecular simulation allows us to predict the orientation of the porphyrin plane in the albumin structure, which supports the kinetic observations.

EXPERIMENTAL PROCEDURES

Materials and Prepration. The synthetic heme, 2-[8{N-(2-methylimidazolyl)}octanoyloxymethyl]-5,10,15,20tetrakis(o-pivalamido)phenylporphinatoiron(II) (FeP) was prepared according to our previously reported procedures (Tsuchida et al., 1995). A recombinant human serum albumin (rHSA; 25 wt %) was obtained from WelFide Corporation (Sumi et al., 1993). The rHSA-FeP solution

10.1021/bc000016e CCC: $19.00 © 2000 American Chemical Society Published on Web 09/30/2000

CO- and O2-Binding to Human Serum Albumin-Heme Hybrid

Bioconjugate Chem., Vol. 11, No. 6, 2000 773

was prepared by the following procedure. Aqueous ascorbic acid (120 mM, 10 µL) was added to an ethanol solution of Fe(III)P (60 µM, 20 mL) under a CO atmosphere to reduce the central ferric ion of FeP. The obtained sixcoordinate carbonyl Fe(II)P in ethanol was slowly added to the phosphate buffer solution (30 mM, pH 7.3) of rHSA (3.75 µM, pH 7.3, 40 mL) through the wall of the round flask. The mixture was then dialyzed with a cellulose membrane against phosphate buffer (pH 7.3) for 2 h and another 16 h at 4 °C. The ethanol content was finally reduced to less than 95 ppm, affording an aqueous solution of rHSA-FeP(8) [[FeP]: 20 µM, FeP/rHSA ) 8 (mol/mol)]. Laser Flash Photolysis. CO and O2 bindings to FeP were expressed by kL on

FeP + L {\ } FeP-L L koff

(1)

Figure 1. Absorption decay of CO rebinding to rHSA-FeP(8) after the laser flash photolysis at 25 °C. The kinetics was composed of three phases and the relaxation curve was fitted by triple-exponentials.

[KL ) kLon/kLoff] RESULTS AND DISCUSSION

where L is CO or O2. The association rate constants of these gaseous ligands (kLon) were obtained from the rebinding kinetics following photodissociation of CO or O2 by laser flash photolysis [Continium Surelight I-10 (Nd:YAG laser SHG: 532 nm, 6 ns pulse width)]. In particular, a CO/O2 competitive binding technique was 2 employed for the determination of kO on (Tsuchida et al., 1993; Collman et al., 1983; Traylor et al., 1985). The Xenon lamp (150 W) was used as the monitor light source. The time dependence of the absorption changes that accompanied the CO or O2 recombination were detected by a Hamamatsu Photonics R636 photomultiplier, and the relaxation curves were fitted to tripleexponentials. A KOFLOC Gas Blender GB-3C prepared the mixture gas with the desired partial pressure. For the solubility of CO and O2 in water at 25 °C, we used the values of 0.94 and 1.25 mM, respectively. Gas concentrations were always at least 10 times that of the FeP concentration, therefore, the pseudo-first-order approximation could be applied throughout. The cuvette of the sample ([FeP]: 20 µM) was fixed into a thermostated holder and the temperature was mainteined from 10 to 35 °C for the Arrhenius equation in order to obtain the activation energy parameters. Binding Equilibria. The gaseous-binding affinity (partial pressure at half binding for FeP, PL1/2 ) 1/[KL]) was determined by spectral changes at various partial pressures of CO or O2 as in previous reports (Tsuchida et al., 1993; Collman et al., 1983). The FeP concentrations of 20 µM were normally used for the UV-vis absorption spectroscopy. The UV-vis absorption measurements were performed using a Shimadzu V-570 spectrometer within the range of 350-700 nm. Molecular Simulations. The orientation of FeP in the hydrophobic cavity of rHSA was predicted by the ESFF force field simulation using an Insight II system (Molecular Simulations Inc.) on a Silicon Graphics O2 computer. First, the domain for FeP was preliminarily predicted using the domain analysis program. The initial structure in which FeP was placed in the crevice between IB and IIA, namely the hemin site, was proposed. The potential energy was obtained after the energy minimization with 100 steps of the conjugate gradient method assuming a dielectric constant of 1.0. Random numbers automatically produced the degrees of freedom of FeP and the residues within the length of 12 Å from the porphyrin center were permitted to move.

CO-Binding Kinetics. We have shown earlier that a maximum of eight FeP molecules are incorporated into certain domains of human serum albumin with binding constants from a K1 of 1.2 × 106 to a K8 of 1.3 × 104 M-1 (Tsuchida et al., 1997, 1999). Three of the eight sites were estimated to be subdomains IB-IIA (hemin site), IIA (bilirbin site), and IIIA (typical palmitic acid site), respectively. For convenience, the eight binding sites of FeP are now named the first site to the eighth site in order of their magnitudes of the binding constants (K1-K8). As a matter of course, the microenvironment around the each FeP in albumin should not be identical. Therefore, one can expect the multiple binding processes of CO and O2 with this synthetic hemoprotein. Flash photolysis experiments were then carried out to characterize the CO- and O2-binidng behavior of rHSA-FeP in detail. First, an aqueous solution of rHSA incorporating eight carbonyl-FePs [rHSA-FeP(8)], which displayed absorption maxima at 427 and 539 nm, was irradiated. The time dependence of the absorption change [∆A(t)] at 443 nm after the laser flash photolysis showed nonexponential kinetics (Figure 1), which was independent of the monitoring wavelength and the laser pulse intensity. On the basis of the careful inspection of the relaxation curve, it could be fitted to triple-exponentials using eq 2,

∆A(t) ) C1 exp(-k1t) + C2 exp(-k2t) + C3 exp(-k3t) (2) where k1, k2, and k3 are apparent rate constants for the each reaction. The fastest component with a k1 of 2.4 × 105 s-1 was relatively minor (C1: 0.16) and found to be independent of the CO concentration. This kinetics should be related to an axial base elimination (Geibel et al., 1978), so that the latter biphasic decay corresponds to the direct rebinding process of CO to the five-Ncoordinated FeP in rHSA (deoxy form). The k2 and k3 values yield the association rate constants for the CO fast and slow phases [kCO on (fast), kon (slow)] (Table 1); CO 5 -1 -1 the kon (slow) (6.7 × 10 M s ) became one-seventh of 6 -1 -1 kCO s ). Interestingly, the absorpon (fast) (4.9 × 10 M tion decay during the CO recombination of rHSA-FeP(1) was almost monophasic (kon ) 4.7 × 106 M-1 s-1). Therefore, it can be supposed that the CO-association rate of FeP is largely affected by the protein environments surrounding the metal center of FeP, e.g., steric hindrance of the amino acid residues and difference in polarity.

774 Bioconjugate Chem., Vol. 11, No. 6, 2000

Komatsu et al.

Table 1. Association and Dissociation Rate Constants of CO and O2 to rHSA-FeP in Phosphate Buffer (pH 7.3) at 25 °C CO 10-6

kon(fast) (M-1 s-1)

rHSA-FeP(2) rHSA-FeP(4) rHSA-FeP(8) Hb(T-state)Ra

4.5 [69] 4.7 [65] 4.9 [73] 0.22

O2 10-6

kon(slow) (M-1 s-1) 0.65 [31] 0.66 [35] 0.67 [27]

10-7

kon(fast) (M-1 s-1)

10-7

kon(slow) (M-1 s-1)

3.4 [67] 3.2 [63] 3.4 [73] 0.29

10-2 koff(fast) (s-1)

10-2 koff(slow) (s-1)

7.4 7.2 7.5 1.8

2.0 2.2 2.0

0.94 [33] 1.0 [37] 0.95 [27]

a In brackets (%); the percentage of the fast and slow components in CO- or O -rebinding to the five-N-coordinated FeP in rHSA, which 2 is estimated from the amplitude of ∆A(t)0). b pH 7, 20 °C (Sawicki et al., 1977; Steinmeier et al., 1975).

Table 2. Free-Energy Changes and Activation Parameters of the CO Association to rHSA-FeP(8) in Phosphate Buffer (pH 7.3) at 25 °C

rHSA-FeP(8) FeP (in toluene)

phase

-1 s-1) 10-6 kCO on (M

∆G‡ (kJ mol-1)

∆H‡ (kJ mol-1)

∆S‡ (J mol-1 K-1)

fast slow

4.9 0.67 6.9

43 49 44 (:∆GqI )

19 23 13

-80 -83 -104

We then calculated the concentration ratio of the fast and slow phases (n) from the initial amplitude after the laser pulse [∆A(t)0)]. For the rHSA-FeP(8), the n value was approximately three, indicating two of the eight FePs exhibited the slow rate constants [kCO on (slow)]. The absorption decays of rHSA-FeP(2) and rHSA-FeP(4) also obeyed the triple-exponentials in which the second and third components again showed the clear CO-concentration dependence. The determined CO kCO on (fast) and kon (slow) were all the same as those of rHSA-FeP(8). On the basis of the n values of rHSA-FeP with different numbers of FeP/rHSA [1, 2, 4, 8 (mol/mol)], it takes into account that two of the eight FePs with the slow rate constant reside in the second and fifth binding sites of the FePs in the albumin structure, i.e., “slow site”. Of course, since the numbers of FePs bound represent average numbers, we made two assumptions; (1) the binding numbers of FeP in an rHSA are identical to the mixture ratio, and (2) in the case of rHSA-FeP(4), for example, the two states (I, II) exist in the solution; I state in which FePs occupy the first to the fourth binding sites (66%), and II state in which FePs occupy the first to the third binding sites and the fifth site (34%). To interpret the reaction profile, the activation parameters and free energy changes (∆Gq) of the CO rebinding to rHSA-FeP(8) were measured (Table 2). The Arrhenius plots of each relaxation component showed a straight line

Figure 2. Double-barrier model for CO rebinding to rHSAFeP. ∆GqI and ∆GqII refer to the top of barriers I and II relative to the reactants. The overall reaction rate is determined by ∆GqI .

in the temperature range of 15-35 °C. ∆Gq of the slow phase (49 kJ mol-1) was significantly higher than that of the fast phase (43 kJ mol-1), and its enthalpy term mainly contributes to the destabilization of the transition state. This result suggests that an additional steric hindrance opposes the binding of CO. In a protein, the multiple free energy barriers generally regulate the entering or leaving of the gaseous ligand. We then proposed a double-barrier model in which CO encounters two barriers on its way from water to the binding site of FeP (Figure 2). A is the FeP-CO bound state, S is the dissociated state and B, is an intermediate state that is only transiently populated. The external barrier II is attributed to the matrix disturbance of the free diffusion of CO from the outer aqueous phase. Once within the pocket of the serum albumin, CO has to overcome energy barrier I for the bond formation. In general, the association rate constants of CO with synthetic hemes and hemoproteins are low compared with those of O2 or NO and far below the diffusion limit at room temperature (Austin et al., 1975; Sharma et al., 1976; Tetreau et al., 1990). Therefore, the overall reaction rates of CO with rHSA-FeP should be governed by the height of barrier I. The ∆Gq of the CO binding to FeP in homogeneous toluene solution was determined to be 44 kJ mol-1, which is identical to that of the fast component of rHSA-FeP(8) [∆Gq(fast): 43 kJ mol-1]. At the “fast sites”, the rHSA structure does not interfere with the CO recombination. In contrast, the height of the barrier increased at the slow site and the CO rebinding could be opposed by the steric hindrance of the bulky amino acid residues located over the porphyrin plane. O2-Binding Kinetics. The O2-binding kinetics of rHSA-FeP showed exactly the same trend observed in the CO recombination. The absorption decay [∆A(t)] of rHSA-FeP(8) following the O2 photodissociation was again fitted by triple-exponentials. The fastest relaxation with the apparent rate constant of k1 (less than 10%) was independent of the O2 concentration. This reaction is considered to be the unfavorable base elimination (Geibel et al., 1978). The slopes of the linear plots of k2 and k3 versus [O2] yield two association rate constants for the O2 2 fast and slow phases [kO on (fast), kon (slow)] (Table 1). The O2 2 differences in the kon (fast) and kO on (slow) values are significantly smaller than that observed in CO. As predicted, the O2-binding kinetics of rHSA-FeP(1) obeyed a single-exponential (kon ) 3.2 × 107 M-1s-1). The n values for rHSA-FeP [FeP/rHSA ) 2, 4, 8 (mol/mol)] were the same as those of the CO recombination. Conse-

CO- and O2-Binding to Human Serum Albumin-Heme Hybrid

Bioconjugate Chem., Vol. 11, No. 6, 2000 775

Table 3. CO- and O2-Binding Affinities of rHSA-FeP in Phosphate Buffer (pH 7.3) at 25 °C CO

rHSA-FeP(4) rHSA-FeP(8)

O2

10-1 P1/2(fast) (Torr)

10-1 P1/2(slow) (Torr)

P1/2(fast) (Torr)

P1/2(slow) (Torr)

1.1 1.2

1.1 1.2

13 14

13 13

quently, the second and the fifth binding sites of FeP are again assigned to the slow sites for O2 binding. CO- and O2-Binding Affinities. The CO- and O2binding affinities (P1/2 is the gaseous pressure at half ligand-binding for FeP) of rHSA-FeP were determined based on the UV-vis absorption spectral changes by the CO and O2 titrations. According to the results of the kinetics experiments, the P1/2 values were divided by two components using eq 3,

Y ) A(K(fast)[L]/(1 + K(fast)[L])) + B(K(slow)[L]/(1 + K(slow)[L])) (3) where Y is total CO and O2 saturation of FeP in rHSA, K(fast) ) [P1/2(fast)]-1, K(slow) ) [P1/2(slow)]-1, and A/B ) n. The obtained P1/2(fast) and P1/2(slow) of the CO and O2 bindings to rHSA-FeP(4) and rHSA-FeP(8) did not show any significant difference (Table 3). Furthermore, the O2 dissociation rate constants in the slow sites [koff(slow)] became smaller than those of the fast sites (Table 1). Since the O2-binding affinities were constant, the dissociation of O2 from the dioxygenated FeP is also retarded in the slow sites. Molecular Simulations. The three-dimensional structure of these synthetic hemoproteins has not yet been solved, although we have attempted to prepare its single crystal. Molecular simulation, however, allows us to predict the orientation of the guest molecule in rHSA. If the CO- and O2-binding plane of FeP, i.e., the fourpivalamido substituted face of the porphyrin, looks toward the albumin surface and there is no steric

hindrance of the protein matrix above, the association rates should not be disturbed. This is actually observed in the fast site. The most stable structure of rHSA-FeP, in which FeP was placed in the subdomains IB-IIA (hemin site), has been simulated by the ESFF force field. The first position of FeP was preliminary predicted using the domain analysis program (see experimental). In the converged state with the lowest potential energy, the four-pivalamido face turns to the outside (Figure 3). In addition, there seems to be a pathway for the small gaseous ligands from the bulk water to the porphyrin plane. This site could be the fast site for the CO and O2 bindings to FeP. These simulation results preliminary support the experimental data based on the kinetic observations. In conclusion, the CO- and O2-binding kinetics of the recombinant human serum albumin incorporating eight tetraphenylporphinatoiron(II) derivatives are composed of three phases, in which the long-lived biphasic decay corresponds to the direct recombination of CO and O2 to the five-N-coordinated FePs in rHSA. Two of the eight sites of FeP (the second and fifth binding sites) are estimated to be the slow sites for the CO and O2 associations. A thermodynamic study revealed that the large ∆Gq of the slow phase in the CO recombination is brought about by the enthalpic factor. The association of the gaseous ligands to the porphyrin in the slow site is probably interfered by the steric hindrance of the amino acid residues of the protein interior. Furthermore, the predicted structure of the rHSA-FeP hybrid by molecular simulation supports the experimental results from the kinetic observations. This procedure can presumably be useful for understanding the CO- and O2binding behaviors on a shorter time scale. ACKNOWLEDGMENT

This work was partially supported by the Core Research for Evolutional Science and Technology, JST, Health Science Research Grants (Artificial Blood Project) of the Ministry of Health and Welfare, Japan, and a Grant-in-Aid for Scientific Research (11650935) form the Ministry of Education, Science, Culture and Sports, Japan. LITERATURE CITED

Figure 3. Molecular simulation image of the most stable conformation of dioxygenated rHSA-FeP(1) in which FeP is placed into the well-known hemin site. The backbones and amino acid residues within the length of 12 Å from the porphyrin center are permitted to move and colored blue. The four-pivalamido groups on the porphyrin ring plane obviously face the outside bulk water.

(1) Ansari, A., Berendzen, J., Bowne, S. F., Frauenfelder, H., Iben, J. E. T., Sauke T. B., Shyamsunder, E., and Young, R. D. (1985) Protein States and Protein Quakes. Proc. Natl. Acad. Sci. U.S.A. 82, 5000-5004. (2) Austin, R. H., Beeson, K. W., Eisenstein, L., Faruenfelder, H., and Gunsalus, I. C. (1975) Dynamics of Ligand Binding to Myoglobin. Biochemistry 14, 5355-5373. (3) Case, D. A., and Karplus, M. (1979) Dynamics of Ligand Binding to Heme Proteins. J. Mol. Biol. 132, 343-368. (4) Collman, J. P., Brauman, J. I., Iverson, B. L., Sessler, J. L., Morris, R. M., and Gibson, Q. H. (1983) O2 and CO Binding to Iron(II) Porphyrins: A Comparison of the “Picket Fence” and “Pocket” Porphyrins. J. Am. Chem. Soc. 105, 3052-3064. (5) Jongeward, K. A., Magde, D., Taube, D. J., Marsters, J. C., Traylor, T. G., and Sharma, V. S. (1988) Picosecond and Nanosecond Geminate Recombination of Myoglobin with CO, O2, NO, and Isocyanate. J. Am. Chem. Soc. 110, 380-387. (6) Geibel, J., Cannon, J., Campbell, D., and Traylor, T. G. (1978) Model Compounds for R-State and T-State Hemoglobins. J. Am. Chem. Soc. 100, 3575-3585. (7) Gibson, Q. H. (1970) The Reaction of Oxygen with Hemoglobin and the Kinetic Basis of the Effect of Salt on Binding of Oxygen. J. Biol. Chem. 245, 3285-3288.

776 Bioconjugate Chem., Vol. 11, No. 6, 2000 (8) Komatsu, T., Hamamatsu, K., Wu, J., and Tsuchida, E. (1999) Physicochemical Properties and O2-Coordination Structure of Human Serum Albumin Incorporating Tetrakis(opivalamido)phenylporphyrinatoiron(II) Derivatives. Bioconjugate Chem. 10, 82-86. (9) Kottalam, J., and Case D. A. (1988) Dynamics of Ligand Escape from the Heme Pockets of Myoglobin. J. Am. Chem. Soc. 110, 7690-7697. (10) Olson, J. S., Sadersen, M. E., and Gibson, Q. H. (1971) The Dissociation of the First Oxygen Molecule from Some Mammalian Oxyhemoglobin. J. Biol. Chem. 246, 5919-5923. (11) Sato, F., Shiro, Y., Sakaguchi, Y., Suzuki, T., Iizuka, T., and Hayashi, H. (1990) New Transient Species of Sperm Whale Myoglobin in Photodissociation of Dioxygen from Oxymyoglobin. J. Biol. Chem. 265, 2004-2010. (12) Sawicki, C. A., and Gibson G. H. (1977) Properties of the T State of Human Oxyhemoglobin Studied by Laser Photolysis. J. Biol. Chem. 252, 7538-7547. (13) Sharma, V. S., Schmidt, M. R., and Ranney, H. M. (1976) Dissociation of CO from Carboxyhemoglobin. J. Biol. Chem. 251, 4267-4272. (14) Steinmeier, R. C., Parkhurst, L. J. (1975) Kinetic Studies on the Five Principle Components of Normal Adult Human Hemoglobin. Biochemistry 14, 1564-1573. (15) Sumi, A., Ohtani, W., Kobayashi, K., Ohmura, T., Yokoyama, K., Nishida, M., and Suyama, T. (1993) Purification and Physicochemical Properties of Recombinant Human Serum Albumin. Biotechnology of Blood Proteins (C. Rivat and J.-F. Stoltz, Ed.) Vol. 227, pp 293-298, John Libbey Eurotext (Montrouge). (16) Tetreau, C., Momentau, M., and Lavalette, D. (1990) Influence of Distal Steric Hindrance on Multiple Energy Barriers in Ligand Binding to Heme Model Compounds. Inorg. Chem. 29, 1727-1731.

Komatsu et al. (17) Traylor, T. G., Tsuchiya, S., Campbell, D., Mitchell, M., Stynes, D., and Koga, N. (1985) Anthracene Heme Cyclophanes. Steric Effects in CO, O2, and RNC Binding. J. Am. Chem. Soc. 107, 604-614. (18) Tsuchida, E., Komatsu, T., Arai, K., and Nishide, H. (1993) Synthesis and Dioxygen-Binding Properties of Doble-sided Porphyrinatoiron(II) Complexes Bearing Covalently Bound Axial Imidazole. J. Chem. Soc., Dalton Trans. 1993, 24652469. (19) Tsuchida, E., Komatsu, T., Kumamoto, S., Ando, K., and Nishide, H. (1995) Synthesis and O2-Binding Properties of Tetraphenylporphyrinatoiron(II) Derivatives Bearing a Proximal Imidazole Covalently Bound at the β-pyrrolic Position. J. Chem. Soc., Perkin Trans. 2 1995, 747-753. (20) Tsuchida, E., Ando, K., Maejima, H., Kawai, N., Komatsu, T., Tekeoka, S., and Nishide, H. (1997) Properties of and Oxygen Binding by Albumin-Tetraphenyl-porphinatoiron(II) Derivative Complexes. Bioconjugate Chem. 8, 534-538. (21) Tsuchida, E., Komatsu, T., Matsukawa, Y., Hamamatsu, K., and Wu, J. (1999) Human Serum Albumin Incorporating Tetrakis(o-pivalamido)phenylporphinatoiron(II) Derivative as a Totally Synthetic O2-Carrying Hemoprotein. Bioconjugate Chem. 10, 797-802. (22) Tsuchida, E., Komatsu, T., Hamamatsu, K., Matsukawa, Y., Tajima, A., Yoshizu, A., Izumi, Y., and Kobayashi, K. (2000) Exchange Transfusion with Albumin-Heme as an Artificial O2-Infusion into Anesthetized Rats: Physiological Responses, O2-Derivery, and Reduction of the Oxidized Hemin Sites by Red Blood Cells. Bioconjugate Chem. 11, 46-50.

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