JP2007302569A - Recombinant human serum albumin-metalloporphyrin complex, artificial oxygen carrier containing the same and erythrocyte substitute - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a recombinant human serum albumin-metalloporphyrin complex capable of adjusting its oxygen affinity. <P>SOLUTION: This recombinant human serum albumin-porphyrin metal complex is obtained by coordinate-bonding a metalloporphyrin with the recombinant human serum albumin to which histidines capable of coordinate-bonding with the metalloporphyrin are introduced by substituting 142 isoleucine, 185 leucine, 138 tyrosine, 115 leucine and/or 139 leucine in the sub-domain IB which is a hem-bonding site of the human serum albumin by a gene recombinant technology, also introducing a key amino acid at another position from the above and further substituting 161 tyrosine with another hydrophobic amino acid different from the tyrosine. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a recombinant human serum albumin-metal porphyrin complex, and an artificial oxygen carrier and artificial red blood cell substitute containing the recombinant human serum albumin-metal porphyrin complex.

  Human serum albumin (HSA) is a simple protein having a molecular weight of 66,500 distributed in many places such as blood, skin, and intercellular fluid in the human body, and is composed of 585 amino acids including 17 disulfide bonds. ing. In addition to maintaining colloid osmotic pressure in blood, it plays an important role in transporting, storing, and distributing various endogenous substances and exogenous drugs. Although research on albumin has been conducted for a long time, it was in 1989 that the entire three-dimensional structure was revealed by X-ray crystal structure analysis (Non-patent Document 1). Thereafter, in 1998, Kary et al. Elucidated the crystal structure of an albumin-fatty acid complex in which seven molecules of fatty acid were bound (Non-patent Document 2). HSA is composed of three domains (I, II, III), which are further composed of two subdomains (A, B).

  If serum albumin can be given oxygen transporting ability such as hemoglobin (Hb), its usefulness as an artificial oxygen carrier (replacement of red blood cells) is extremely high. For example, it is widely used for lifesaving measures in the emergency medical field. There is no doubt that it will greatly contribute to the medical welfare of mankind.

In plasma, there is also a glycoprotein called hemopexin that spontaneously incorporates hemin (iron (III) protoporphyrin) and constitutes a heme protein. Since hemin liberated from oxidized Hb (meth Hb) is cytotoxic, it is quickly captured by this hemopexin and transported to the liver (Non-patent Document 3). The hemin molecule incorporated into hemopexin is very strongly fixed by an axial coordination bond with two histidine residues and a multiple hydrogen bond with amino acid residues (K> 10 ¹² M ⁻¹ ) (non-patented) Reference 4). However, since the blood concentration of hemopexin is as low as <17 μM, when it is saturated, serum albumin substitutes for its function (the binding constant of albumin and hemin is about 10 ⁸ M ⁻¹ , see Non-Patent Document 5). . Most recently, Carter et al. And our research group independently succeeded in X-ray crystal structure analysis of albumin-hemin-myristic acid complex (Non-patent Documents 6 and 7). Hemin is incorporated into the subdomain IB of albumin, the axial coordination bond with 161 tyrosine, and electrostatic between three basic amino acid (lysine 190, histidine 146, arginine 114) residues and the propionic acid side chain It is fixed by interaction. The molecular environment surrounding the hemin binding site is composed only of hydrophobic amino acid residues and has structural similarity to the heme pocket of myoglobin (Mb). When a sodium dithionite aqueous solution is added to an HSA-hemin complex aqueous solution under a nitrogen atmosphere and hemin is reduced to an iron (II) protoporphyrin complex (protoheme), the visible absorption spectrum shows iron (II) coordinated with 161 tyrosine. Forms a five-coordinate high spin complex. However, even if oxygen is passed therethrough, the central iron (II) is instantly oxidized and a stable oxygen complex cannot be obtained (Non-patent Document 8).

  On the other hand, the research group of the present inventors found that albumin obtained by efficiently including iron (II) tetraphenylporphyrin in which four hydrophobic substituents and an axial base ligand are covalently bonded in the molecule was included in albumin. -It has been clarified that the heme complex can adsorb and desorb oxygen in the same manner as Hb and Mb under physiological conditions (physiological saline, pH 7.3, 37 ° C) (Patent Documents 1 and 2). In addition, the research group of the present inventors analyzed in detail the electronic state of the oxygen coordination complex of albumin-heme complex from infrared absorption, resonance Raman, magnetic circular dichroism spectrum, and the like (Non-Patent Document 9). And animal experiments have demonstrated quantitatively that the albumin-heme complex functions as a serum heme protein capable of transporting oxygen even in vivo.

  However, synthetic hemes acting as oxygen-coordinating active centers in these albumin-heme complexes are iron (II) having a special steric structure in which four hydrophobic substituents and an axial base ligand are covalently bonded in the molecule. ) Limited to tetraphenylporphyrin. It goes without saying that this type of heme derivative synthesis method has multiple steps, not only is it difficult to mass-produce, but also the metabolic degradation process of the synthetic heme derivative after in vivo administration remains unclear. There were many current situations. If an albumin-metal porphyrin complex obtained by immobilizing natural metal porphyrin, for example, iron (II) protoporphyrin, or a derivative thereof on serum albumin can express oxygen coordination ability similar to Hb and Mb, Biocompatibility and productivity are greatly improved, and it is considered to be an extremely useful material.

  Therefore, the present inventors introduced a histidine that acts as a proximal base into the subdomain IB that is the porphyrin-binding site of albumin by gene recombination technology without covalently bonding the axial base into the metalloporphyrin molecule. For example, the imidazole group of the histidine is axially coordinated to the metal porphyrin, and a five-coordinate complex is obtained, and oxygen coordination complexes found in Hb and Mb should be formed by aeration of oxygen therethrough. I thought.

  Carter and colleagues prepared a recombinant albumin-protoheme complex in which protoheme was coordinated by introducing a hydrophilic amino acid (histidine or glutamine) into human serum albumin by genetic recombination technology, and examined its oxygen binding reaction (Patent Document 3). However, protoheme is the only oxygen coordination active center used, and quantitative analysis of oxygen coordination ability such as oxygen binding constant has not been performed. Furthermore, our research group has shown that, based on the results of research on artificial oxygen carriers using synthetic heme for many years, when a hydrophilic group exists in the vicinity of the oxygen coordination site, the central metal (II) that mediates protons. It has been demonstrated that the oxidation reaction of oxygen is promoted and the stability of the oxygen complex decreases (Non-patent Document 10).

  Under such circumstances, our research group introduced histidine acting as a proximal base into subdomain IB, which is the porphyrin-binding site of albumin, by means of genetic recombination technology, and further, oxygen coordination locus If 161 tyrosine bound to the side is replaced with a hydrophobic amino acid by gene recombination technology and an artificial hem pocket is constructed, a metal porphyrin complex is axially coordinated and stable oxygen complex In addition to protoheme, the active center is thought to be able to be universalized by a wide variety of metalloporphyrins, and a recombinant human serum albumin-metalloporphyrin complex was prepared to reversibly bind oxygen. First revealed (Non-Patent Document 8, Patent Document 4).

  However, when practically using these aqueous solutions of recombinant human serum albumin-metalloporphyrin complexes as artificial oxygen carriers, oxygen binding parameters such as oxygen affinity should be adapted to the respective use conditions and clinical indications. It will be necessary to adjust. In the case of the metal tetraphenylporphyrin having a complex steric structure having the above-described four hydrophobic substituents and an axial base ligand in the molecule, by adjusting the chemical structure of the substituent structure and the axial base ligand, It has been clarified that the oxygen affinity can be adjusted (Non-patent Document 10).

That is, regarding the recombinant human serum albumin-metal porphyrin complex, establishment of a new technique for adjusting the oxygen coordination ability has been awaited.
DC Carter et al., Science 244: 1195-1198 (1989) S. Curry et al., Nature Struct. Biol. 5: 827-835 (1998) E. Tolosano et al., DNA Cell Biol., 21: 297-306 (2002) M. Paoli et al., Nature Struct. Biol. 6: 926-931 (1999) PA Adams et al., Biochem. J. 191: 95-102 (1980) M. Wardell et al., Biochem. Biophys. Res. Commun. 291: 813-819 (2002) PA Zunszain et al., BMC Struct. Biol. 3: 6 (2003) T. Komatsu et al., J. Am. Chem. Soc. 127: 15933-15942 (2005) E. Tsuchida et al., Bioconjugate Chem. 10: 797-802 (1999) T. Komatsu et al., Bull. Chem. Soc. Jpn. 74: 1695-1702 (2001) JP-A-8-301873 JP 2003-40893 A Special Table 2002-200282 JP 2006-45172 A

  Therefore, the present invention eliminates the above-mentioned problems of the prior art, and can provide a recombinant human serum albumin-metal porphyrin complex that can adjust oxygen affinity, and an artificial oxygen carrier containing this recombinant albumin-metal porphyrin complex. And to provide a red blood cell substitute.

  An important requirement for imparting oxygen coordination ability to a metalloporphyrin is that imidazole as an axial base ligand is bound to the fifth coordination site of the porphyrin center metal. On the other hand, oxygen affinity is greatly influenced by the molecular environment in the vicinity of the sixth coordination site to which oxygen is bound. In the case of Mb which is a natural heme protein, the N atom of histidine 64 located at a distance of 2.77 to 2.95 km from the oxygen atom of the coordination oxygen molecule forms a hydrogen bond with the coordination oxygen via the proton, It contributes to stabilization of oxygen complexes and increased oxygen affinity (SE Phillips and BP Schoeborn, Nature 292: 81-82 (1981)). Springer et al. Synthesized recombinant Mb in which Mb histidine 64 was converted to other amino acids using gene recombination technology, analyzed its oxygen coordination ability in detail, and developed natural histidine 64. It was clarified that the oxygen affinity was highest when the structure was formed (BA Springer et al., Chem. Rev. 94: 699-714 (1994)).

  As a result of intensive studies on molecular environment design and functional expression for controlling the oxygen affinity of the recombinant human serum albumin-metalloporphyrin complex, the present inventors have determined that subdomain IB, which is the porphyrin-binding site of albumin, By introducing histidine that acts as a proximal base of metalloporphyrin by gene recombination technology, and introducing a key amino acid at a position different from that (particularly at a position where it can interact with coordinated oxygen), Hb It has been found that the oxygen affinity complex of the recombinant human serum albumin-metalloporphyrin complex can be controlled by adjusting the position and structure of the key amino acid. It came to be completed.

  That is, according to the first aspect of the present invention, in subdomain IB that is a heme binding site of human serum albumin, histidine coordinated with metalloporphyrin is transformed into 142 isoleucine and 185 leucine of albumin by gene recombination technology. 138 tyrosine, 115 leucine or 139 leucine is introduced, and at least one key amino acid is introduced by a genetic recombination technique at a position different from the introduction position, and 161 tyrosine is a hydrophobic amino acid 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 protein is provided.

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

  According to a third aspect of the present invention, there is provided a red blood cell substitute containing the recombinant human serum albumin-metal porphyrin complex of the present invention. In this case, the metal porphyrin has iron (II) or cobalt (II) as a central metal.

  In the recombinant human serum albumin-metal porphyrin complex according to the present invention, the metal porphyrin is bound to the inside of albumin by the axial coordination bond of the central metal to histidine introduced into the subdomain IB that is a porphyrin binding site by gene recombination technology. In addition to forming a five-coordinate high-spin complex that is anchored to oxygen and forming a highly stable oxygen-coordination complex by oxygen aeration, a key amino acid is introduced in the vicinity of the coordinating oxygen by genetic recombination technology. Therefore, it has the characteristic that oxygen affinity can be adjusted. Furthermore, the recombinant human serum albumin of the present invention can fix not only protoheme, which is the oxygen binding site of Hb, but also synthetic metalloporphyrins through the axial base coordination to express oxygen binding ability. In addition, oxygen affinity can be controlled by selecting the type and position of the key amino acid. Moreover, the artificial oxygen carrier containing the recombinant human serum albumin-metal porphyrin complex of the present invention can be used as an alternative to highly safe blood for transfusion even when administered in vivo. In addition, the recombinant human serum albumin-metalloporphyrin complex of the present invention is an extracorporeal circuit such as a preservation solution of a transplanted organ or tissue, a tissue culture solution, an anticancer therapeutic sensitizer for a tumor, a preoperative blood dilution solution, an artificial cardiopulmonary lung, etc. Can be used as a filling fluid, transplanted organ perfusion fluid, oxygen supply fluid to ischemic sites (myocardial infarction, cerebral infarction, respiratory failure, etc.), chronic anemia treatment agent, liquid ventilation reflux fluid, gas adsorbent, It is also useful as a redox catalyst, oxygen oxidation reaction catalyst, and oxygen addition reaction catalyst.

  The albumin-metal porphyrin complex of the present invention introduces a histidine coordinated with a metal porphyrin into a predetermined position in a subdomain IB, which is a heme binding site, by a genetic recombination technique, and a key amino acid at another position. Can be obtained by binding metal porphyrin by axial coordination to recombinant human serum albumin in which 161 tyrosine is substituted with a hydrophobic amino acid other than tyrosine.

  The recombinant human serum albumin used has 142 isoleucine, 185 leucine, 138 tyrosine, 115 leucine or 139 leucine replaced with histidine in subdomain IB (histidine introduced at any one of these positions is a metalloporphyrin). The key amino acid is introduced at a position different from the introduction position (these key amino acids are coordinating amino acids, and histidine is actually introduced among the introduction positions of the histidine). When introduced at a position other than the position indicated, it may be axially coordinated to the metal porphyrin), and 161 tyrosine is a non-coordinating hydrophobic amino acid substituted recombinant albumin.

  The key amino acid is preferably a polar amino acid, particularly asparagine, glutamine, lysine, histidine, arginine or proline. In addition, glycine, alanine, valine, leucine, isoleucine, proline, tryptophan, or phenylalanine can be suitably used as a non-coordinating hydrophobic amino acid other than histidine that substitutes 161 tyrosine.

  The metal porphyrin is preferably metal protoporphyrin, metal deuteroporphyrin, metal diacetyl deuteroporphyrin, metal mesoporphyrin, metal diformyl porphyrin, metal tetraphenylporphyrin, metal octaethyl porphyrin. And what is its central metal? Iron or cobalt, particularly iron (II) or cobalt (II) is preferred.

  A surface-modified recombinant serum albumin-metal porphyrin double complex can be obtained by covalently bonding a polyethylene glycol group to the molecular surface of the recombinant human serum albumin-metal porphyrin complex of the present invention. Preferably, a polyethylene glycol group is introduced by a thioether bond, and in particular, the amino group of a lysine residue present on the surface of serum albumin is converted to a thiolate group with iminothiolane, and then reacted with polyethylene glycol having a terminal maleimide group. Thus, a surface-modified recombinant serum albumin-metal porphyrin complex obtained by introducing a polyethylene glycol group through a thioether bond is desirable.

  Since the recombinant human serum albumin-metal porphyrin complex of the present invention can bind and dissociate oxygen like Hb and Mb in water, it functions as a fully synthetic artificial oxygen carrier. Of course, when administered into the body, it not only serves as a substitute for red blood cells, but also stores organs or tissues suitable for transplantation in an aqueous solution of this recombinant albumin-metal porphyrin complex, so that It enables safe and long-term storage of organs or tissues. In addition, this recombinant human serum albumin-metal porphyrin complex can act as a culture medium that promotes tissue growth, and if it is administered to the hypoxic site of a tumor, it can pass through fine capillaries where red blood cells cannot enter. The tumor hypoxic site can be oxygenated, and then the tumor can be reduced or cured by irradiating the affected area with radiation immediately.

  Artificial oxygen carriers can be used for resuscitation of hemorrhagic shock (replacement of blood for transfusion), preoperative blood dilution, extracorporeal circulation such as cardiopulmonary bypass, perfusion of transplanted organs, ischemia Oxygen supply fluid to the site (myocardial infarction, cerebral infarction, respiratory failure, etc.), chronic anemia treatment, liquid ventilation fluid, use for rare blood type patients, response to transfusion refusal patients for religious reasons Application to animal medicine is expected. The artificial oxygen carrier is obtained by dispersing the recombinant human serum albumin-metal porphyrin complex of the present invention in physiological saline. The concentration of the recombinant human serum albumin-metal porphyrin complex varies depending on its use, but as a red blood cell substitute, it can be used at a heme concentration of about 9.2 mM, and at other concentrations.

  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 the artificial oxygen carrier.

  The recombinant human serum albumin of the present invention has a substitution or insertion of histidine in the subdomain IB, a substitution or insertion of a key amino acid at a different position, and a non-coordinating hydrophobicity of 161 tyrosine. Obtained by substitution with an amino acid. Introduction of an amino acid at a specific position is accomplished by various conventional means currently known to achieve such recombination in albumin. Furthermore, the preparation of the recombinant albumin of the present invention can be carried out 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, the recombinant albumin can be produced using a conventional culture method using yeast. For example, mutagenesis can be performed using the Quick Change XL Site-Directed Mutagenesis Kit (STRATAGENE), and expression can be performed using the Pichia Expression Kit (Invitrogen), CE Peterson et al., Biochemistry, 36, 7012. -7017 (1997). The resulting recombinant human serum albumin can be purified by conventional methods, for example, column chromatography packed with Blue Sepharose 6 Fast Flow, followed by column chromatography packed with SUPERDEX pg75.

  Modification of the molecular surface of the recombinant human serum albumin-metal porphyrin complex with a polyethylene glycol group can be carried out, for example, by the method described in JP-A-2006-45173. The average number of polyethylene glycol bonds per albumin molecule is preferably 1 to 15, and the average molecular weight of polyethylene glycol is preferably 2,000 to 5,000.

  The recombinant human serum albumin-metal porphyrin complex of the present invention should be prepared by the usual method described in, for example, the aforementioned T. Komatsu et al., J. Am. Chem. Soc. 127: 15933-15942 (2005). Can do. If the metal porphyrin is in the form of an iron (III) complex, an appropriate reducing agent (sodium dithionite, ascorbic acid, etc.) can be used to reduce the central metal from trivalent to divalent by a conventional method. , Oxygen binding activity can be imparted. In any case, when it comes into contact with oxygen, a stable oxygen complex is rapidly formed. Further, these complexes can adsorb and desorb oxygen according to the oxygen partial pressure. This oxygen bond dissociation can be repeated reversibly and acts as an oxygen carrier.

  In addition to oxygen, in the case of a gas that is coordinated to a metal, a corresponding coordination complex can be formed (for example, carbon monoxide, nitrogen monoxide, nitrogen dioxide, etc.). For these reasons, the recombinant albumin-metal porphyrin complex of the present invention, especially in the case of an iron (II) or cobalt (II) complex, exhibits an effective function for many uses described above as an artificial oxygen carrier. Of course, application as a redox reaction catalyst and gas adsorbent in homogeneous and heterogeneous systems is possible.

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

Example 1
For the purpose of observing the oxygen binding reaction of the recombinant human serum albumin-metal porphyrin complex of the present invention, 142 isoleucine of human serum albumin is replaced with histidine, 161 tyrosine is replaced with leucine, and 185 leucine is replaced with asparagine. Substituted recombinant human serum albumin (I142H / Y161L / L185N) was produced by conventional site-directed mutagenesis and conventional culture using Pichia yeast. That is, mutagenesis was performed using Quick Change XL Site-Directed Mutagenesis Kit (STRATAGENE), expression was performed using Pichia Expression Kit (Invitrogen), CE Peterson et al., Biochemistry, 36, 7012-7017. (1997).

The obtained recombinant human serum albumin (I142H / Y161L / L185N) was purified by column chromatography packed with Blue Sepharose 6 First Flow, followed by column chromatography packed with SUPERDEX pg75. This recombinant albumin (I142H / Y161L / L185N) (20 μM, 50 mM phosphate buffered aqueous solution) to iron (III) protoporphyrin (0.266 mM, DMSO solution) has a porphyrin / albumin molar ratio of 1.1 / 1. And mixed with rotary stirring in the dark for 12 hours. The obtained mixed solution was washed with an ultrafiltration device (ultramolecular weight: 10 kDa), and concentrated and diluted with a 50 mM phosphate buffer aqueous solution repeatedly until the DMSO concentration became 0.1% or less. The ultraviolet-visible absorption spectrum of the obtained aqueous solution is shown in FIG. 1 (shown as Fe ³⁺ ).

This aqueous solution was sufficiently substituted with argon and deoxygenated, and then an aqueous sodium thionite solution was added to reduce the central iron to iron (II), and recombinant human serum albumin (I142H / Y161L / L185N) − An iron (II) protoporphyrin complex was prepared. The UV-visible absorption spectrum of this aqueous solution showed λmax: 425, 532, and 559 nm, and this was very similar to the deoxy-type spectral pattern of Mb, which revealed the formation of a Fe (II) 5-coordinated high spin complex. (FIG. 1, shown as Fe ²⁺ ). It was considered that the imidazole group of 142 histidine was coordinated to central iron as an axial base, and a deoxy form was obtained. When oxygen is bubbled there, it immediately shifts to an oxygen complex type spectrum (λmax: 411, 537, 576 nm) (shown as Fe ²⁺ (O ₂ ) in FIG. 1), and when carbon monoxide is bubbled, stable monoxide is obtained. Carbon complexes (λmax: 419, 537, 564 nm) were obtained (FIG. 1, indicated as Fe ²⁺ (CO)).

Example 2
The recombinant albumin (I142H / Y161L / L185N) -iron (II) protoporphyrin complex aqueous solution prepared in Example 1 was irradiated with a leather flash (ND: YAG laser, 532 nm, pulse width 6 ns), and a non-equilibrium state occurred instantaneously. From the time-resolved analysis of the change in the visible absorption spectrum to the equilibrium state, the oxygen affinity (P ₅₀ ) and the oxygen bond dissociation rate constants (k _on , k _off ) were calculated. Actual measurement was performed by the method described in Komatsu et al., J. Am. Chem. Soc. 2005, 176, 30552-30265. As a result, it was found that there are two components in the oxygen binding reaction, and two orientation structures of iron (II) protoporphyrin in the heme pocket were found (type I and type II). Type I P ₅₀ at 1 Torr (22 ° C.), recombinant albumin (I142H / Y161L) having no L185N - iron (II) as low as 1/18 compared to protoporphyrin complex (oxygen affinity be 18 times higher Is close to the value of Mb. k _on is 1.3 × 10 ⁷ M ⁻¹ s ⁻¹ , k _off is 21 s ⁻¹ , and these values are also Mb values (k _on : 1.4 × 10 ⁷ M ⁻¹ s ⁻¹ , k _off : similar to 12 s ⁻¹ ). Also, P ₅₀ of the II type is 14Torr (22 ℃), it was 27 times higher than the value of Mb (oxygen affinity will be 1/27 low). This was presumed to be due to a large k _off value (290 s ⁻¹ ) based on the distortion of the axial base ligand.

Example 3
In Example 1, a recombinant human serum albumin (I142H / Y161F / L185N) in which 161 tyrosine of human serum albumin was replaced with phenylalanine instead of leucine was subjected to the conventional site-directed mutagenesis and the conventional method using Pichia yeast. A recombinant human serum albumin (I142H / Y161F / L185N) -iron (II) protoporphyrin complex was prepared in the same manner except that it was produced by a culture method. The UV-visible absorption spectrum of this aqueous solution showed λmax: 425, 532, and 559 nm, and this was very similar to the deoxy-type spectral pattern of Mb, which revealed the formation of a Fe (II) 5-coordinated high spin complex. . It was considered that the imidazole group of 142 histidine was coordinated to central iron as an axial base. When oxygen is passed there, it immediately shifts to an oxygen complex type spectrum (λmax: 411, 540, 579 nm), and when carbon monoxide is passed, stable carbon monoxide complexes (λmax: 419, 539, 566 nm) are obtained. It was.

Example 4
In Example 2, instead of the recombinant human serum albumin (I142H / Y161L / L185N) -iron (II) protoporphyrin complex, the recombinant human serum albumin (I142H / Y161F / L185N) -iron (II) protoporphyrin complex is used. Except for the above, oxygen affinity (P ₅₀ ) and oxygen bond dissociation rate constants (k _on , k _off ) were calculated in exactly the same manner. 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 ₅₀ is 2.3 Torr (22 ° C.), it was 4.5 times higher than in Mb (oxygen affinity is 1 / 4.5 lower).

Example 5
In Example 1, instead of replacing human serum albumin 185 leucine with asparagine, recombinant human serum albumin (1822H / Y161L / L182H) with 182 leucine replaced with histidine was used for conventional site-directed mutagenesis and Pichia yeast. A recombinant human serum albumin (I142H / Y161L / L182H) -iron (II) protoporphyrin complex was prepared according to the same procedure except that the complex was produced by the conventional culture method. The UV-visible absorption spectrum of this aqueous solution showed λmax: 426, 532, and 557 nm, and this was very similar to the deoxy-type spectral pattern of Mb, which revealed the formation of a Fe (II) 5-coordinated high spin complex. . It was considered that the imidazole group of 142 histidine was coordinated to central iron as an axial base. When oxygen is bubbled there, it immediately shifts to an oxygen complex type spectrum (λmax: 412, 540, 576 nm), and when carbon monoxide is bubbled, stable carbon monoxide complexes (λmax: 419, 539, 565 nm) are obtained. It was.

Example 6
In Example 1, recombinant human serum albumin (I142H / Y161F / R186H) in which 185 arginine was replaced with histidine instead of 185 leucine in human serum albumin with asparagine, and phenylalanine was substituted with 161 tyrosine in place of leucine Is produced by conventional site-directed mutagenesis and a conventional culture method using Pichia yeast according to the same procedure, except that recombinant human serum albumin (I142H / Y161F / R186H) -iron (II) protoporphyrin complex Was prepared. The UV-visible absorption spectrum of this aqueous solution showed λmax: 426, 533, and 558 nm, and this was very similar to the deoxy-type spectrum pattern of Mb, which revealed the formation of a Fe (II) 5-coordinated high spin complex. . It was considered that the imidazole group of 142 histidine was coordinated to central iron as an axial base. When oxygen is bubbled there, it immediately shifts to an oxygen complex type spectrum (λmax: 411, 541, 777 nm), and when carbon monoxide is bubbled, a stable carbon monoxide complex (λmax: 419, 538, 566 nm) is obtained. It was.

Example 7
In Example 1, recombinant human serum albumin (I142H / Y161V / L182R) in which 161 tyrosine of human serum albumin was replaced with valine instead of leucine and 182 leucine was replaced with arginine instead of 185 leucine with asparagine Was produced by conventional site-directed mutagenesis and a conventional culture method using Pichia yeast according to the same procedure, except that recombinant human serum albumin (I142H / Y161V / L182R) -iron (II) protoporphyrin complex Was prepared. The UV-visible absorption spectrum of this aqueous solution showed λmax: 427, 534, and 557 nm, and this was very similar to the deoxy-type spectrum pattern of Mb, which revealed the formation of a Fe (II) 5-coordinated high spin complex. . It was considered that the imidazole group of 142 histidine was coordinated to central iron as an axial base. When oxygen is bubbled there, it immediately shifts to an oxygen complex type spectrum (λmax: 411, 540, 576 nm), and when carbon monoxide is bubbled, stable carbon monoxide complexes (λmax: 419, 537, 565 nm) are obtained. It was.

Example 8
In Example 1, the human serum albumin 142 isoleucine was replaced with histidine instead of histidine, 161 tyrosine was replaced with alanine instead of leucine, and 185 leucine was replaced with histidine instead of asparagine Recombinant human serum albumin (I142K / Y161A) was prepared according to the same procedure except that recombinant human serum albumin (I142K / Y161A / L185H) was produced by conventional site-directed mutagenesis and a conventional culture method using Pichia yeast. / L185H) -iron (II) protoporphyrin complex was prepared. The UV-visible absorption spectrum of this aqueous solution showed λmax: 426, 535, and 557 nm, and this was very similar to the deoxy-type spectral pattern of Mb, which revealed the formation of a Fe (II) 5-coordinated high spin complex. . It was considered that the imidazole group of 185 histidine was coordinated to central iron as an axial base. When oxygen is bubbled there, it immediately shifts to an oxygen complex type spectrum (λmax: 412, 541, 578 nm), and when carbon monoxide is bubbled, a stable carbon monoxide complex (λmax: 419, 536, 568 nm) is obtained. It was.

Example 9
Iminothiolane (manufactured by Pierce Chemical Co., Ltd., iminothiolane / albumin) was synthesized in the phosphate buffer solution (pH 7.0) of (I142H / Y161L / L185N) -iron (III) protoporphyrin complex (albumin concentration: 0.1 wt%) synthesized in Example 1. 20 (mol / mol)) was added and the mixture was slowly stirred at room temperature for 4 hours. Subsequently, a small excess molar one-side maleimide one-end methyl-polyethylene glycol (Sun Bright Memar 50-H, Mw: 5,000, manufactured by NOF Corporation) is added to albumin and reacted for 2 hours under the same conditions. It was. The obtained mixture was repeatedly concentrated and washed with 1 L of an aqueous phosphate buffer solution using an ultrafiltration device (manufactured by Advantech, UHP-76K, ultramolecular weight membrane: 5 kDa), and adjusted to a desired concentration. The obtained red surface-modified recombinant human serum albumin-iron (III) porphyrin complex aqueous solution was passed through a 0.45 μm sterilization filter (manufactured by Advantech, DISMIC 25CS045AS) for final adjustment.

  When the molecular weight was measured by the matrix-assisted laser desorption / ionization mass spectrometry (MALDI-TOFMS) (manufactured by Shimadzu Corporation, KRATOS AXIMA-CFR) of the obtained surface-modified recombinant human serum albumin-iron (III) porphyrin complex The number of polyethylene glycol chains bound to the albumin surface was found to be 6 on average.

  This aqueous solution was sufficiently replaced with argon and deoxygenated, and then an aqueous sodium nitrite solution was added to reduce the central iron to iron (II), and surface-modified recombinant human serum albumin (I142H / Y161L / L185N) − An iron (II) protoporphyrin complex was prepared. The UV-visible absorption spectrum of this aqueous solution showed λmax: 425, 532, and 559 nm, and this was very similar to the deoxy-type spectral pattern of Mb, which revealed the formation of a Fe (II) 5-coordinated high spin complex. . It was considered that the imidazole group of 142 histidine was coordinated to central iron as an axial base, and a deoxy form was obtained. When oxygen is bubbled there, it immediately shifts to an oxygen complex type spectrum (λmax: 412, 537, 576 nm), and when carbon monoxide is bubbled, a stable carbon monoxide complex (λmax: 419, 536, 564 nm) is obtained. It was.

Example 10
In Example 1, recombinant human serum albumin (I142H / Y161L / L185N) -iron (II) due to the same procedure except that iron (III) deuteroporphyrin was used instead of iron (III) protoporphyrin. A teloporphyrin complex was prepared. The ultraviolet-visible absorption spectrum of this aqueous solution shows λmax: 413, 517, and 546 nm, which is very similar to the deoxy-type spectral pattern of Mb, and thus the formation of a Fe (II) 5-coordinated high spin complex is clarified. It was. It was considered that the imidazole group of 142 histidine was coordinated to central iron as an axial base, and a deoxy form was obtained. When oxygen is bubbled there, it immediately shifts to an oxygen complex type spectrum (λmax: 401, 528, 563 nm), and when carbon monoxide is bubbled, a stable carbon monoxide complex (λmax: 409, 528, 556 nm) is obtained. It was.

Example 11
Recombinant human serum albumin (I142H / Y161L / L185N) -cobalt (II) protoporphyrin was prepared in the same manner as in Example 1 except that cobalt (II) protoporphyrin was used instead of iron (III) protoporphyrin. A complex was prepared. The ultraviolet-visible absorption spectrum of this aqueous solution showed λmax: 406, 558 nm, which was very similar to the deoxy-type spectral pattern of cobalt Mb, and thus the formation of a Co (II) 5-coordinated high spin complex was clarified. . It was considered that the imidazole group of 142 histidine was coordinated to central iron as an axial base, and a deoxy form was obtained. When oxygen was bubbled there, it immediately shifted to an oxygen complex type spectrum (λmax: 426, 539, 578 nm).

UV-visible absorption spectra of recombinant human serum albumin-iron protoporphyrin complex in various forms obtained in Example 1.

Claims (8)

  In subdomain IB, which is the heme-binding site of human serum albumin, histidine that coordinates with metalloporphyrin replaces 142 isoleucine, 185 leucine, 138 tyrosine, 115 leucine or 139 leucine of albumin by genetic recombination technology. Recombinant human serum albumin introduced with at least one key amino acid introduced by genetic recombination technology at a position different from the introduction position and 161 tyrosine substituted with a hydrophobic amino acid other than tyrosine, metalloporphyrin Recombinant human serum albumin-porphyrin metal complex coordinated with.   The recombinant human serum albumin-metal porphyrin complex according to claim 1, wherein the key amino acid is a polar amino acid.   The metal porphyrin is at least one selected from the group consisting of metal protoporphyrin, metal deuteroporphyrin, metal diacetyl deuteroporphyrin, metal mesoporphyrin, metal diformyl porphyrin, metal tetraphenylporphyrin and metal octaethylporphyrin. The recombinant human serum albumin-metal porphyrin complex according to claim 1, which is a metal porphyrin.   The recombinant human serum albumin-metal porphyrin according to any one of claims 1 to 3, wherein a polyethylene glycol group is covalently bonded to the molecular surface of the recombinant human serum albumin-metal porphyrin complex. Complex.   The recombinant human serum albumin-metal porphyrin complex according to any one of claims 1 to 4, wherein the metal is iron or cobalt.   6. The recombinant human serum albumin-metal porphyrin complex according to claim 5, wherein the metal is iron (II) or cobalt (II).   An artificial oxygen carrier containing the recombinant human serum albumin-metal porphyrin complex according to claim 6.   An erythrocyte substitute containing the recombinant human serum albumin-metal porphyrin complex according to claim 6.

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