WO2006016560A1

WO2006016560A1 - Recombinant human serum albumin-metallic phorphyrin complex, artificial oxygen carrier and erythrocyte substitute

Info

Publication number: WO2006016560A1
Application number: PCT/JP2005/014532
Authority: WO
Inventors: Eishun Tsuchida; Teruyuki Komatsu; Naomi Ohmichi; Stephen Curry
Original assignee: Eishun Tsuchida; Teruyuki Komatsu; Naomi Ohmichi; Stephen Curry
Priority date: 2004-08-09
Filing date: 2005-08-08
Publication date: 2006-02-16
Also published as: JP2006045172A

Abstract

A recombinant human serum albumin-metallic porphyrin complex wherein a metallic porphyrin is bonded, via a coordinate bond, to a recombinant human serum albumin having at least one histidine residue capable of binding to the 5-coordination site of metallic porphyrin, which has been transferred into the sub domain IB (a hem binding site) of human serum albumin by a gene recombination technique, and having a substitution of tyrosine at the 161-position by a hydrophobic amino acid other than tyrosine.

Description

Specification

Recombinant human serum albumin-metal borphyrin complex, artificial oxygen carrier and erythrocyte substitute

Technical field

[0001] The present invention relates to a recombinant human serum albumin-metal borphyrin complex, and an artificial oxygen carrier and a red blood cell substitute containing the recombinant human serum albumin. In particular, the present invention relates to a complex of recombinant human serum albumin and metallophyllin in which histidine and a hydrophobic amino acid are introduced into subdomain IB, which is the heme binding site of human serum albumin, by genetic recombination technology, and the complex. Related to artificial oxygen carriers and red blood cell substitutes.

Background art

[0002] Human serum albumin (HSA) is a prosthetic molecule with a molecular weight of about 66,500 distributed in many places in the body, including blood and intercellular fluid, and a simple protein without sugar chains. The 585 amino acid power including the disulfide bond is also constructed. Since it accounts for about 60% of blood protein, its colloid osmotic pressure plays a role in maintaining water balance inside and outside the blood vessel and maintaining vascular circulation. In addition, since it has the ability to bind non-specific multimolecules, it plays an important physiological role in the transport, storage, distribution and metabolism of various endogenous substances. There are reports of research on albumins of various animal species, and the amino acid sequence of mammalian albumin such as ushi and rat has a structural homology of over 60% with human serum albumin. The study of albumin has been conducted for a long time, and the entire three-dimensional structure was revealed by force X-ray crystallographic analysis in 1989 (DC Carter et al., Science 244: 1195-1198 (1989)). Later, in 1998, Curry et al. Elucidated the crystal structure of an albumin-fatty acid complex in which seven molecules of fatty acids were bound (S. Curry et al., Nature Struct. Biol. 5, 827-835 ( 1 998)). HSA consists of three domains (1, II, III), which are further composed of two subdomains (A, B).

[0003] If serum albumin can be given oxygen transport ability such as hemoglobin (Hb) Therefore, the value of clinical use as an artificial oxygen carrier (red blood cell substitute) is extremely high. For example, even if it is considered to be applied to a lifesaving measure in an emergency medical field, its great effect can be easily estimated. Interestingly, some plasma proteins incorporate hemin (iron (III) protoporphyrin) into heme proteins. Hemin released from metHb into the bloodstream generates cytotoxicity and reactive oxygen, so it is quickly captured by a glycoprotein called hemopexin and transported to the liver (E. Tolosano et al., DNA Cell Biol, 21: 2 97-306 (2002)) From the results of X-ray crystallographic analysis of the hemopexin monohemine complex, the hemin molecule has an axial coordination with two histidine residues and amino acids. Multiple hydrogen bonds with residues have been shown to be very strongly immobilized (K> 10 ¹² M ^_1 ) (M. Paoli et al., Nature Struct. Biol. 6: 926— 931 (1999)). However, the blood concentration of hemopexin is as low as 17 M, so if this is saturated, serum albumin will replace that function. The binding constant between albumin and ^hemin has been reported to be about 10 ⁸ ^M_1 (PA Adams et al., Biochem. J. 191: 95-102 (1980)). Most recently, Carter et al. And the inventors independently succeeded in analyzing the crystal structure of albumin-hemin-myristic acid complex (M. Wardell et al., Biochem. Biophys. Res. Commun. 291: 813-819). (2002), PA Zunszain et al., BMC Struct. Biol. 3: 6 (2003)). Hemin is incorporated into the subdomain IB of albumin, and is coordinated with 16 1 tyrosine and electrostatic between three basic amino acid (lysine 190, histidine 146, arginine 114) residues and the propionate side chain. Fixed by dynamic interaction. The molecular environment surrounding the hemin binding site is composed solely of hydrophobic amino acid residues and is similar to the heme pocket of myoglobin (Mb). Under a nitrogen atmosphere, add sodium nithionite aqueous solution to this aqueous solution, and then add hemin to iron (

II) When reduced to a protoporphyrin complex (protoheme), the visible absorption spectrum forms an iron (11) 5-coordinated high-spin complex coordinated with 161 tyrosin. However, even though oxygen was vented there, central iron (II) was instantly oxidized, and it was difficult to obtain a stable oxygen complex.

On the other hand, the research group of the present inventors found that iron (II) tetrafluoro-porphyrin having four hydrophobic substituents and having an axial base ligand covalently bound in the molecule is effective for hydrophobic interaction with albumin. The resulting albumin heme complex is capable of adsorbing and desorbing oxygen in the same way as Hb and myoglobin (Mb) under physiological conditions (physiological saline, pH 7.3, 37 ° C). (Japanese Unexamined Patent Publication No. 8-301873, Japanese Unexamined Patent Publication No. 2003-40893). In addition to detailed analysis of the electronic state of oxygen coordination complexes from infrared absorption, resonance Raman, magnetic circular dichroism spectra, etc., animal experiments show that it functions as a serum heme protein that can carry oxygen in vivo. (E. Tsuchida et al, Bioconjugate Chem. 11: 46-50 (2000)). In addition, hypertension (side effects), which is a drawback of molecular Hb preparations that are currently being developed as artificial red blood cells, has not been observed at all (E. Tsuch ida et al., J. Biomed. Mater. Res. 64A: 257—261 (2003)). Since albumin generates electrostatic repulsion between negative charges with the basement membrane surrounding the endothelial cells, which has a lower isoelectric point than Hb, the mono-acidic acid is a vascular endothelial relaxing factor whose vascular endothelial permeability is low.匕 Do not erase nitrogen! Because there is no sudden fluctuation in blood pressure.

[0005] However, synthetic hem, which functions as an oxygen coordination active center in these albumin monoheme complexes, has a special three-dimensional structure with four hydrophobic substituents and an axial base ligand in the molecule. It must be iron (II) tetraporphyrin, which is quite different from iron (II) protoporphyrin in natural Hb. In other words, to date, the structural requirement for conferring oxygen coordination ability to the iron (フィ) porphyrin of albumin-hemium complex is to first hold at least a proximal base covalently linked in the molecule. It was. For this reason, the synthesis process is multi-stage and has the disadvantages that mass production is difficult. In addition, there are still many unclear points regarding the metabolic degradation process after administration to the body. If natural metalloporphyrin, such as iron (II) protoporphyrin, or its derivatives, albumin metalporphyrin complex power prepared by immobilizing serum albumin, can express oxygen coordination ability similar to Hb and Mb, Its usefulness is immeasurable. In other words, the establishment of a new technology that achieves reversible oxygen bond release such as Hb with an albumin metal volfilin complex in which metal (II) porphyrin with no proximal base introduced into the molecule is bound to albumin has been awaited. I was angry.

Recently, Carter et al. Prepared a recombinant albumin-protoheme complex in which a hydrophilic amino acid (histidine or glutamine) was introduced into human serum albumin by genetic recombination technology, and protoheme was coordinated, and its oxygen-binding reaction. (Special Table 2002-50086 2). However, protoheme is the only oxygen-coordinating active center used. Quantitative analysis of oxygen coordination ability, such as the coupling constant, has not been made, and a stable oxygen complex was actually obtained.

Disclosure of the invention

[0007] Accordingly, the present invention solves the above-mentioned problems of the prior art and can form a stable oxygen complex by introducing a metal porphyrin having no proximal base in the molecule. It is an object of the present invention to provide a metal borphyrin complex, and an artificial oxygen carrier and erythrocyte substitute containing the albumin metal complex.

[0008] The most important condition for imparting oxygen coordination ability to a metalloporphyrin is that imidazole serving as an axial base ligand is bound to the fifth coordination site of iron (鉄) porphyrin. The inventors of the present invention have designed a molecular environment and developed a function for allowing imidazole, which is a proximal base, to be axially coordinated to the fifth coordination position without introducing an axial base into the metalloporphyrin molecule through a covalent bond. As a result of extensive research, when histidine acting as a proximal base is introduced into subdomain IB, which is the porphyrin-binding site of albumin, by genetic recombination technology, the imidazole group of the histidine is axially coordinated with metal porphyrin. Thus, a pentacoordinate complex was obtained, and further oxygen aeration was considered to enable the formation of oxygen coordination complexes found in Hb and Mb. However, as a result of research on oxygen infusion using synthetic heme for many years, our research group has found that when a hydrophilic group exists in the vicinity of the oxygen coordination site, the central metal (する) that mediates protons. It has been clarified that the oxidation reaction of oxygen is promoted and the stability of the oxygen complex decreases (T. Komatsu et al., Bull. Chem. Soc. Jpn. 74: 1695-1702 (2001)).

Under such a background, the present inventors introduced histidine that acts as a proximal base into subdomain IB, which is a porphyrin-binding site of albumin, by genetic recombination technology, and oxygen By introducing a hydrophobic amino acid into the coordinating locus using genetic recombination technology and constructing an artificial hem pocket, the metal borphyrin complex immobilized inside via a coordinate coordination bond becomes a stable oxygen complex. It was found that the active center can be universalized not only with protoheme but also with a wide variety of metalloporphyrins, thereby completing the present invention.

[0010] That is, according to the first aspect of the present invention, it is a heme binding site of human serum albumin. In subdomain IB, at least one histidine coordinated to the 5-coordination locus of metal porphyrin was introduced by genetic recombination technology, and 161 tyrosine was replaced with a hydrophobic amino acid other than tyrosin. Recombinant human serum albumin-porphyrin metal complex in which metal porphyrin is coordinated to recombinant human serum albumin is provided. V ヽ Needless to say, in this complex, the introduced histidine is coordinated to the pentacoordination site of the metalloporphyrin.

[0011] According to a second aspect of the present invention, there is provided an artificial oxygen carrier (oxygen infusion) containing the recombinant human serum albumin metal porphyrin complex of the present invention. In this case, the metal borphyrin has iron (II) or cobalt (II) as the central metal.

[0012] Further, according to the third aspect of the present invention, there is provided an erythrocyte substitute containing the recombinant human serum albumin-metal porphyrin complex of the present invention. In this case, the metalloporphyrin has iron (II) or cobalt (II) as the central metal.

Brief Description of Drawings

[0013] [Fig. 1] Fig. 1 shows Fe (III), Fe (II) (deoxy), Fe (II) (O) of recombinant human serum albumin (I142HZY161L) -iron (II) borphyrin complex (Oxy), Fe (II) (CO) (

2

It is a graph which shows the visible absorption spectrum change of a carbonyl body. BEST MODE FOR CARRYING OUT THE INVENTION

[0014] The albumin metal-borphyrin complex of the present invention is a group in which at least one histidine is introduced by genetic recombination technology in subdomain IB, which is a heme binding site, and 161 tyrosine is substituted with a hydrophobic amino acid other than tyrosine. It can be obtained by binding metalloporphyrin to human serum albumin by axial coordination. The recombinant human serum albumin used is preferably a combination of 142 isoleucine, 185 leucine, 138 tyrosine, 115 leucine, 139 leucine and / or 182 leucine strength histidine and 161 tyrosine substituted with a hydrophobic amino acid. Replaced human serum albumin. The recombinant human serum albumin may be a recombinant human serum albumin inserted at least one into subdomain IB, which is a heme binding site of human serum albumin. As the hydrophobic amino acid for substituting 161 tyrosine, glycine, alanine, norine, leucine, isoleucine, proline, tributophan or phenylalanine can be preferably used. [0015] The metal porphyrin is preferably a metal protoporphyrin, a metal deuteroporphyrin, a metal diacetyl deuteroporphyrin, a metal mesoporphyrin, a metal diformyl porphyrin, a metal tetraphenyl porphyrin, or a metal otaethyl porphyrin . The central metal is preferably iron or cobalt, particularly iron (II) or cobalt (II).

[0016] The recombinant human serum albumin metal volfilin complex of the present invention can bind and dissociate oxygen like Hb and Mb in water, and thus functions as a fully synthetic oxygen infusion (artificial oxygen carrier). Of course, when administered to the body, this recombinant human serum albumin not only serves as a substitute for red blood cells, but also preserves organs or tissues suitable for transplantation in a solution of the metal borphyrin complex, so that organs prior to transplantation can be stored. Or the organization can be stored safely and for a long time. In addition, this recombinant human serum albumin-metal vorphyrin complex acts as a culture medium that promotes tissue growth, and if administered to the hypoxic site of the tumor, it can pass through fine capillaries where red blood cells cannot enter. Oxygenation of the oxygen site is realized, and then the tumor can be reduced or healed by immediately irradiating the affected area with radiation.

[0017] In addition to resuscitation fluid for hemorrhagic shock (blood substitute for blood transfusion), oxygen transfusion can be used for preoperative blood dilution, extracorporeal circulation such as cardiopulmonary bypass, perfusion fluid for transplanted organs, false Oxygen supply fluid to the blood site (myocardial infarction, cerebral infarction, respiratory failure, etc.), chronic anemia treatment agent, liquid ventilation recirculating fluid, use for rare blood type patients, transfusion rejection patients for religious reasons Is expected to be applied to animal medicine. An artificial oxygen carrier (oxygen infusion) can be obtained by dispersing the recombinant human serum albumin-metal borphyrin complex of the present invention in physiological saline. Recombinant human serum albumin The concentration of metalloflurin complex varies depending on its use, but as a blood substitute (red blood cell substitute), a heme concentration of about 9.2 mMZL can be used, and other concentrations can be used.

[0018] Power! In addition, when the metal porphyrin is, for example, a complex of metal ions belonging to the 4th to 5th periods, the added value as a catalyst for the oxidation-reduction reaction, oxygen oxidation reaction or oxygen addition reaction is also high. Therefore, the porphyrin metal complex of the present invention has characteristics as a gas adsorbent, a redox catalyst, an oxygen oxidation reaction catalyst, and an oxygen addition reaction catalyst in addition to oxygen infusion.

[0019] The recombinant albumin of the present invention generally contains amino acid positions of the subdomain IB listed above. Or histidine is substituted or inserted into the amino acid in the vicinity thereof, or 161 thycin is substituted with a non-coordinating hydrophobic amino acid. Introduction of an amino acid at a specific position is accomplished by a variety of conventional means now known to achieve extensive recombination in albumin. Further, the recombinant albumin of the present invention can be prepared by mutating the DNA encoding albumin using conventional site-directed mutagenesis. This mutation is a small mutation that does not affect the original three-dimensional structure, physical properties, and characteristics of albumin. In addition, genetically modified albumin can be produced using a conventional culture method utilizing yeast. For example, mutagenesis can be performed using the Quick Change XL Site-Directed Mutagenesis Kit (STRATAGENE), and expression can be performed using the Picma Expression Kit (Invitrogen), CE Peterson et al., Bioc hemistry, 36 , 7012-7017 (1997). The obtained recombinant human serum albumin can be purified by a conventional method, for example, column chromatography packed with Blue Sepharose 6 First Flow, followed by column chromatography packed with Cefacryl S200HR.

[0020] The albumin-metal borphyrin complex of the present invention can be prepared by the usual method described in Non-Patent Document 7, for example. If the metalloporphyrin is in the form of an iron (III) complex, use an appropriate reducing agent (sodium nithionite, ascorbic acid, etc.) and reduce the central metal from trivalent to divalent by a conventional method. For example, oxygen binding activity can be imparted. In either case, when it comes into contact with oxygen, a stable oxygen complex is rapidly formed. These complexes can adsorb and desorb oxygen according to the partial pressure of oxygen. This oxygen bond dissociation can be repeated reversibly and acts as an oxygen carrier.

In the case of a gas that is coordinated to a metal other than oxygen, a corresponding coordination complex can be formed (for example, carbon monoxide, nitrogen monoxide, nitrogen dioxide, etc.). For these reasons, the recombinant albumin metal volfilin complex of the present invention exhibits an effective function for many of the above-mentioned indications as an oxygen infusion solution, particularly in the case of an iron (II) or cobalt (II) complex, It can be used as a redox catalyst for homogeneous and heterogeneous systems, and as a gas adsorbent.

Hereinafter, the present invention will be described in detail with reference to examples. The present invention is not limited to the examples. Needless to say, it is not specified.

[0023] Example 1

Recombinant albumin of the present invention Recombinant human serum albumin (I142H / Y161L) in which 142 isoleucine of human serum albumin is replaced with histidine and 161 tyrosine is replaced with leucine for the purpose of observing the oxygen-binding reaction of the metalloflurin complex of the present invention. ) Were produced by conventional site-directed mutagenesis and conventional culture using Pichia yeast. In other words, mutations were introduced using the Quick Change XL Site-Directed Mutagenesis Kit (STRATAGEN E), and expression was performed using the Pichia Expression Kit (Invitrogen).

This was performed according to the method described in Peterson et al., Biochemistry, 36, 7012-7017 (1997). The obtained recombinant human serum albumin (I142HZY161L) was purified by column chromatography packed with Blue Sepharose 6 First Flow, followed by column chromatography packed with Cefacryl S200HR. This recombinant albumin (I142H / Y161L) (20 / z M, 50 mM phosphate buffered aqueous solution) and iron (III) protoporphyrin (0.266 mM, DMSO solution) have a porphyrin Z albumin molar ratio of 1. lZl. The mixture was mixed and mixed for 12 hours with rotary stirring in the dark. The obtained mixed solution was washed with an ultrafiltration device (ultramolecular weight: 10,000), and concentrated and diluted with a 50 mM phosphate buffer aqueous solution repeatedly until the DMSO concentration became 0.1% or less. The magnetic circular dichroism spectra of the recombinant albumin-iron (III) protoporphyrin complex aqueous solution thus prepared showed peaks at 366 (—), 40 2 (+), and 419 (—) nm. Was similar to the spectral pattern of met-Mb.

[0024] After this aqueous solution was sufficiently substituted with argon and deoxygenated, an aqueous sodium thionite solution was added to reduce the central iron to iron (Π), and recombinant human serum albumin (I142HZY 161L ) —Iron (II) protoporphyrin complex was prepared. The UV-visible absorption spectrum of this aqueous solution showed max: 426 and 559 nm, which is very similar to the Mb deoxy-type spectral pattern, which indicates the formation of a Fe (II) 5-coordinated high-spin complex. (Fig. 1). That is, the imidazole group of 142 histidine is coordinated to central iron as an axial base, and it is considered that a deoxy form was obtained. When oxygen is passed through this recombinant human serum albumin (I142HZY161L) monoiron (Π) protoporphyrin complex aqueous solution, the oxygen complex (oxy ) Type spectrum ( _max : 412, 537, 573 nm) and aeration of carbon monoxide yielded a stable carbon monoxide complex (carbonyl) (max: 419, 538, 565 nm) (Fig. 1). (Figure 1 shows Fe (III), Fe (II) (deoxy), Fe (II) (O) (oxy) isomers of recombinant human serum albumin (I142HZY161L) -iron (II) borphyrin complex. , Fe (II) (CO) body

2

(A graph showing changes in the visible absorption spectrum of (carbonyl)).

[0025] Example 2

Recombinant albumin prepared in Example 1 (I142HZY161L) —iron (II) protoporphyrin complex aqueous solution is irradiated with leather flash (Nd: YAG laser, 532 nm, pulse width 6 ns) and equilibrated from the non-equilibrium state that occurs instantaneously. Calculate the bond dissociation rate constants (k, k) and oxygen affinity (P) of oxygen from the time-resolved analysis of the visible absorption spectrum change to the state.

It was. The actual measurement was performed according to the method described in JP Collman et al., J. Am. Chem. Soc. 105, 3052-3064 (19 83), and TG Traylor et al., J. Am. Chem. Soc. 107: 604—6 14 (1985). As a result, it was found that there are two components in the oxygen binding reaction, and two orientation structures of iron (II) protoporphyrin exist in the heme pocket (type I and type II). Type I P is 18 Torr (22 ° C), and the value of Mb is

50

Compared to 35 times larger (oxygen affinity is 1Z35). k is 7.5 X 10 ⁶ M— ¹ and Mb (l. 4 X on

10 ⁷ that there was a value as changed such Chikaratsu of M- ^1), the low oxygen affinity greater k value (221s off

—Mb k is 12 s— and type II P is 134 Torr (22 ° C off 50

It was 262 times greater than the Mb value (oxygen affinity is 1Z262). This is presumed to be due to a larger k value (1.7 X 10 ³ s—) based on the strain of the axial base ligand.

It was.

[0026] Example 3

Instead of substituting human serum albumin 142 isoleucine with histidine for example 1, recombinant human serum albumin (L185HZY161L) in which human serum albumin 185 leucine is replaced with histidine is treated with conventional site-specific A recombinant human serum albumin (L 185H / Y161L) -iron (II) protoporphyrin complex was prepared according to the same procedure except that it was produced by mutagenesis and a conventional culture method using Pichia yeast. The UV-visible absorption spectrum of this aqueous solution shows max: 422, 558 nm, which is a Mb deoxy-type spectrum. The formation of Fe (ll) 5-coordinated high-spin complex was clarified by the similarities to the gold pattern. It is probable that the imidazole group of 185 histidine was coordinated to central iron as an axial base, and a deoxy form was obtained. When oxygen is bubbled through this aqueous complex solution, it immediately shifts to an oxygen complex type spectrum (max: 412, 530, 570 nm), and when carbon monoxide is bubbled, stable carbon monoxide complexes (max: 419, 537, 560 nm). )was gotten.

[0027] Example 4

Recombinant human serum albumin (I142HZY161L) —Iron (Π) deuterovorphiline complex according to the same procedure except that iron (III) deuteroporphyrin was used instead of iron (III) protoporphyrin in Example 1. Was prepared. The UV-visible absorption spectrum of this aqueous solution shows max: 413, 517, and 546 nm, which is very similar to the Mb deoxy-type spectral pattern, so Fe (II) 5-coordinated high spin The formation of the complex became obvious. 1 42 It is probable that the imidazole group of histidine was coordinated to central iron as an axial base, and a deoxy form was obtained. When oxygen is bubbled through this complex aqueous solution, it immediately shifts to an oxygen complex type spectrum ( _max: 401, 528, 563 nm), and when carbon monoxide is bubbled, a stable carbon monoxide complex (λ max: 409 , 528, 556nm) force S was obtained.

[0028] Example 5

A recombinant human serum albumin (I142HZY161L) -cobalt (Π) protoporphyrin complex was prepared in the same manner as in Example 1 except that cobalt (Π) protoporphyrin was used instead of iron (III) protoporphyrin. . The UV-visible absorption spectrum of this aqueous solution shows max: 406 and 558 nm, which is very similar to the cobalt Mb deoxy-type spectral pattern. The formation of the complex became obvious. The imidazole group of 142 histidine was coordinated to central iron as an axial base, and it was considered that a deoxy form was obtained. When oxygen was bubbled through this aqueous complex solution, an immediate shift to an oxygen complex type vector (max: 426, 539, 578 nm) was obtained.

[0029] As described above, the recombinant human serum albumin-metal borphyrin complex according to the present invention is a porphyrin-binding site (sub-site) by gene recombination technology, even for metalloporphyrins that do not hold a proximal base covalently in the molecule. The histidine introduced into domain IB) and the central metal are incorporated into the albumin through axial coordination, and human serum Al Since 161 tyrosine of bumine is substituted with a predetermined hydrophobic amino acid, a 5-coordinate high-spin complex can be formed and a highly stable oxygen-coordinated complex can be formed. In other words, the recombinant human serum albumin according to the present invention fixes not only protohem, which is an oxygen binding site of Hb, but also a synthetic metalloporphyrin through an axial base coordination to express oxygen binding ability. be able to. In addition, the artificial oxygen carrier (oxygen infusion solution) containing the recombinant human serum albumin metal volfilin complex of the present invention can be used as a blood substitute for highly safe blood for transfusion even when administered in vivo. To the transplanted organ or tissue preservation solution, tissue culture solution, tumor anti-cancer treatment sensitizer, preoperative blood dilution, extracorporeal circulation such as cardiopulmonary bypass, perfusion solution of transplanted organ, ischemic site It can be used as an oxygen supply solution (myocardial infarction, cerebral infarction, respiratory failure, etc.), a therapeutic agent for chronic anemia, and a circulating fluid for liquid ventilation. In addition, it is also useful as a gas adsorbent, oxidation-reduction catalyst, oxygen oxidation reaction catalyst, and oxygen addition reaction catalyst.

Claims

The scope of the claims

[1] In subdomain IB, which is the heme-binding site of human serum albumin, at least one histidine coordinated to the 5-coordinate locus of metal borphyrin is introduced by genetic recombination technology, and 161 tyrosine other than tyrosine A recombinant human serum albumin porphyrin metal complex in which a metalloporphyrin is coordinated to a recombinant human serum albumin substituted with a hydrophobic amino acid.

[2] Histidine power of the albumin 142 isoleucine, 185 leucine, 138 tyrosine

The recombinant human serum albumin metal volphiline complex according to claim 1, introduced by substituting 115 leucine, 139 leucine and / or 182 leucine.

[3] The recombinant human serum albumin-metal borphyrin complex according to claim 1, wherein the hydrophobic amino acid is glycine, alanine, parin, leucine, isoleucine, proline, tryptophan or phenylalanin.

[4] The metal porphyrinka, metal protoporphyrin, metal deuteroporphyrin, metal diacetyl deuteroporphyrin, metal mesoporphyrin, metal diformyl porphyrin, metal tetraphenylporphyrin or metal otaethyl porphyrin Item 5. Recombinant human serum albumin metal borphyrin complex according to item 1.

5. The recombinant human serum albumin metal volfilin complex according to claim 1, wherein the metalloporphyrin has iron or cobalt as a central metal.

6. The recombinant human serum albumin metal porphyrin complex according to claim 5, wherein the central metal is iron (II) or cobalt (II).

[7] An artificial oxygen carrier comprising the recombinant human serum albumin-metal borphyrin complex according to claim 6.

[8] An erythrocyte substitute comprising the recombinant human serum albumin-metal borphyrin complex according to claim 6.