JP5413786B2 - Cross-linked structure of protein polymer - Google Patents

Description

  The present invention relates to a structure in which a protein polymer is crosslinked. More specifically, the present invention relates to a structure in which a protein polymer represented by the general formula (IA) or the general formula (IB) or a salt thereof is crosslinked.

  In recent years, structures in which biomolecules are integrated as building blocks have attracted attention as biomaterials. For example, a structure obtained by reacting hemoglobin with glutaraldehyde and polymerizing them is known (see, for example, Patent Documents 1 and 2). This structure has been tried to be applied when treating a mammal suffering from massive blood loss (see, for example, Patent Document 3). A structure obtained by replacing the heme of myoglobin with a heme modified with a long chain and micellizing the myoglobin is known (see, for example, Non-Patent Document 1).

  However, a sponge-like three-dimensional structure of heme protein has not been known.

Japanese National Patent Publication No. 1-501471 JP-T-2006-516994 JP-T-2006-502231

I.C.Reynhout et al., “Self-Assembled Architectures from Biohybrid Triblock Copolymers”, J. Am. Chem. Soc., 2007, 129, p.2327-2332.

  Accordingly, an object of the present invention is to provide a sponge-like three-dimensional structure in which biomolecules are integrated.

  The present invention is a structure in which a protein polymer or a salt thereof is crosslinked, and the protein polymer or a salt thereof is a protein polymer represented by the general formula (IA) or the general formula (IB) or a salt thereof.

In the formula (IA) or (IB),
R ¹ , R ² , R ³ and R ⁴ each independently represents a hydrogen atom, a lower alkyl group, a lower alkyl group or a lower alkenyl group substituted with a halogen atom,
Y is a group represented by -Z1-Y1-Z2-Y2- or -Z1-Y1-, wherein Z1 is a group bonded to -CO- in formula (IA) and formula (IB). Means
Z1 is selected from the group consisting of O, NH and S;
Y1 and Y2 are each independently formula _{- (CH 2) n1 -,} - (C 6 H 4) n5 -, - (CH 2) n1 - (C 6 H 4) n5 -, - (C 6 H _{4) n5 - (CH 2)} n1 -, - (CH 2) n1 - (C 6 H 4) n5 - (CH 2) n2 -, - (CH 2) n1 -O- (CH 2) n2 -, - _{(CH 2) n1 -O- (CH} 2) n2 -O- (CH 2) n3 - or _{- (CH 2) n1 -O- (} CH 2) n2 -O- (CH 2) n3 -O- (CH ₂ ) a group represented by _n4- , wherein n1, n2, n3, n4 and n5 each independently represents an integer of 1 to 20, and the (C ₆ H ₄ ) is one or more substituents May have a group,
Z2 is selected from the group consisting of NHCO, CONH, NH, O, N = N, N = CH, CH = N, C = C;
M is selected from the group consisting of Fe ²⁺ , Fe ³⁺ , Zn ²⁺ , Co ²⁺ , Co ³⁺ , Mn ²⁺ and Mn ³⁺ ,
m and n are each an integer, 1 ≦ m + n ≦ 1000,
X means X in the hemoprotein mutant, and the hemoprotein mutant is represented by the formula (II);

  The mutant is obtained by replacing one amino acid residue with a cysteine residue in the amino acid sequence of a natural heme protein. In the mutant, -SH means a side chain thiol group of the cysteine residue, Heme means natural heme, and formula HS-X means an apo form of the mutant.

  The present invention also relates to a method for producing a structure in which a protein polymer or a salt thereof is cross-linked, wherein the protein polymer represented by the general formula (IA) or the general formula (IB) or a salt thereof is covalently bonded between molecules. And the protein polymer represented by the general formula (IA) or the general formula (IB) or a salt thereof is crosslinked between molecules to obtain the structure.

In the formula (IA) or (IB),
R ¹ , R ² , R ³ and R ⁴ each independently represents a hydrogen atom, a lower alkyl group, a lower alkyl group or a lower alkenyl group substituted with a halogen atom,
Y is a group represented by -Z1-Y1-Z2-Y2- or -Z1-Y1-, wherein Z1 is a group bonded to -CO- in formula (IA) and formula (IB). Means
Z1 is selected from the group consisting of O, NH and S;
Y1 and Y2 are each independently formula _{- (CH 2) n1 -,} - (C 6 H 4) n5 -, - (CH 2) n1 - (C 6 H 4) n5 -, - (C 6 H _{4) n5 - (CH 2)} n1 -, - (CH 2) n1 - (C 6 H 4) n5 - (CH 2) n2 -, - (CH 2) n1 -O- (CH 2) n2 -, - _{(CH 2) n1 -O- (CH} 2) n2 -O- (CH 2) n3 - or _{- (CH 2) n1 -O- (} CH 2) n2 -O- (CH 2) n3 -O- (CH ₂ ) a group represented by _n4- , wherein n1, n2, n3, n4 and n5 each independently represents an integer of 1 to 20, and the (C ₆ H ₄ ) is one or more substituents May have a group,
Z2 is selected from the group consisting of NHCO, CONH, NH, O, N = N, N = CH, CH = N, C = C;
M is selected from the group consisting of Fe ²⁺ , Fe ³⁺ , Zn ²⁺ , Co ²⁺ , Co ³⁺ , Mn ²⁺ and Mn ³⁺ ,
m and n are each an integer, 1 ≦ m + n ≦ 1000,
X means X in the hemoprotein mutant, and the hemoprotein mutant is represented by the formula (II);

  The mutant is obtained by replacing one amino acid residue with a cysteine residue in the amino acid sequence of a natural heme protein. In the mutant, -SH means a side chain thiol group of the cysteine residue, Heme means natural heme, and formula HS-X means an apo form of the mutant.

  The structure of the present invention is a sponge-like three-dimensional structure having a hemoprotein, which is a biomolecule, as a building block.

FIG. 1a shows the UV-vis spectrum of natural myoglobin. FIG. 1 b shows the UV-vis spectrum of the protein polymer (I-13) produced in Example 1. FIG. 1c shows a UV-vis spectrum of an oxygen complex of natural myoglobin. FIG. 1 d shows the UV-vis spectrum of the oxygen complex of the protein polymer (I-13) produced in Example 1. FIG. 2 a shows size exclusion chromatography on the protein polymer (I-13) obtained in Example 1. FIG. 2b shows size exclusion chromatography. The solid line shows the size exclusion chromatography for the crosslinked structure obtained in Example 1, and the broken line shows the protein polymer (I-13) obtained in Example 1. FIG. 2c shows size exclusion chromatography. The solid line shows the size exclusion chromatography for the crosslinked structure obtained in Example 1, and the broken line shows the protein polymer (I-13) obtained in Example 1. FIG. 3 shows a size exclusion chromatogram of the protein polymer (I-13) obtained in Example 1 measured under conditions where the concentration was changed. FIG. 4 a shows the entire AFM image of the protein polymer (I-13) obtained in Example 1. FIG. 4b shows the height profile of the AFM along the white line in FIG. 4a. FIG. 5 a shows an ESI-TOF-MS spectrum of the protein monomer (V-13) obtained in Example 1. FIG. 5b shows a deconvoluted ESI-TOF-MS spectrum of the protein monomer (V-13). FIG. 6 shows the result of electrophoresis in a denatured state of the crosslinked structure obtained in Example 1. FIG. 7 a shows an SEM image of the crosslinked structure obtained in Example 1. FIG. 7 b shows an SEM image of the crosslinked structure obtained in Example 1. FIG. 7 c shows an SEM image of the crosslinked structure obtained in Example 1. FIG. 8 a shows the UV-vis spectrum of the crosslinked structure obtained in Example 1. FIG. 8 b shows the UV-vis spectrum of the oxygen complex of the crosslinked structure obtained in Example 1.

  As described above, the present invention is a structure in which a protein polymer or a salt thereof is crosslinked, and the protein polymer or a salt thereof is a protein polymer or a salt thereof represented by the general formula (IA) or (IB). is there.

  The present invention also provides a method for producing a structure in which a protein polymer or a salt thereof is crosslinked as described above, wherein the protein polymer or a salt thereof represented by the general formula (IA) or (IB) And the protein polymer represented by the general formula (IA) or (IB) or a salt thereof is crosslinked between molecules to obtain the structure.

  In the present invention, the protein polymer represented by the general formulas (IA) and (IB) includes both a random copolymer and a block copolymer, and is not limited to either one.

  In the production method of the present invention, the condition for forming the covalent bond is preferably a condition for generating a radical. Conditions for generating this radical are contact with peracid such as hydrogen peroxide, peracetic acid, metachloroperbenzoic acid, cumene hydroperoxide, X-ray irradiation, electron beam irradiation, ion beam irradiation, gamma ray irradiation, light (UV) It is preferably one or more selected from the group consisting of irradiation.

  Moreover, this invention is a biological device containing the structure of this invention.

  In the present invention, “lower” means 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, unless otherwise specified.

  Examples of the “lower alkyl group” in the “lower alkyl group” and the “lower alkyl group substituted with a halogen atom” include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, neopentyl, hexyl and the like. Such a linear or branched alkane residue having 1 to 6 carbon atoms is meant. Preferable examples of the lower alkyl group include alkyl having 1 to 5 carbon atoms. Preferred examples of the alkyl group having 1 to 5 carbon atoms include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, neopentyl and the like.

  As the “lower alkenyl group”, vinyl, allyl, isopropenyl, 1-, 2- or 3-butenyl, 1-, 2-, 3- or 4-pentenyl, 1-, 2-, 3-, 4- or Examples thereof include straight-chain or branched alkenyl groups having 2 to 6 carbon atoms such as 5-hexenyl. Preferred lower alkenyl groups include vinyl.

  Examples of the “halogen atom” include fluorine, chlorine, bromine, iodine, etc. Among them, fluorine is preferable.

  An example of “a lower alkyl group substituted with a halogen atom” means one or more hydrogen atoms of the lower alkyl group substituted with the halogen atom. Preferable examples of the lower alkyl group substituted with the halogen atom include methyl iodide, dichloromethyl, trichloromethyl, trifluoromethyl and the like. A preferred lower alkyl group substituted with a halogen atom is trifluoromethyl.

  m and n are each an integer, and 1 ≦ m + n ≦ 1000. Preferably, 10 ≦ m + n ≦ 100, more preferably 30 ≦ m + n ≦ 100.

As Y1 and _{Y2, - (C 6 H 4} ) n5 -, - (CH 2) n1 - (C 6 H 4) n5 -, - (C 6 H 4) n5 - (CH 2) n1 -, - (CH _{2) n1 - (C 6 H} 4) n5 - (CH 2) n2 - it may be represented by, as described above _{in, (C} 6 _{H 4)} may have one or more substituents. When n5 is 2 or more, (C ₆ H ₄ ) may each independently have one or more substituents. Examples of the substituent include a hydroxy group, a cyclic, linear or branched aliphatic saturated or unsaturated hydrocarbon group, an alkoxy group, an acyloxy group, an alkoxycarbonyloxy group, an amino group, an alkylamino group, an arylamino group, An aryl group, a nitro group, an oxo group, a halogen atom, etc. are mentioned.

  In the formula (IA) and formula (IB), when Y is represented by -Z1-Y1-Z2-Y2-, Y is, for example, No. represented by the following table. 1-No. There are 42 formulas.

  When Y is represented by -Z1-Y1-Z2-Y2-, Y represents No. in Table 1 above. 1-6, 10-15, 19-26, 31-38 are preferable. 1, 2, 3, 10, 11, 12, 19, 20, 21, 22, 31, 32, 33, 34 are more preferable.

  In the formula (IA) and formula (IB), when Y is represented by -Z1-Y1-, Y is, for example, No. represented by the following table. 43-No. There are 51 formulas.

  When Y is represented by -Z1-Y1-, No. Groups represented by the formulas 43 to 48 are preferred. Groups represented by the formulas 43 to 45 are more preferable.

  The polymers and salts thereof in the present invention may take the form of solvates, which are also included in the scope of the present invention. Preferred solvates include hydrates and ethanol solvates.

In the protein polymer represented by the formula (IA) or (IB) or a salt thereof, the natural heme protein is not particularly limited as long as it is a protein having one or more heme. For example, cytochrome, hemoglobin, myoglobin, or peroxidase Is mentioned. Examples of the cytochrome include cytochrome b ₅₆₂ , cytochrome b ₅ and cytochrome P450 _CAM , and among them, cytochrome b ₅₆₂ is preferable. Examples of the hemoglobin include hemoglobin (human), hemoglobin (bovine), hemoglobin (equine), and hemoglobin (rat). Among them, hemoglobin (human) is preferable. Examples of the myoglobin include myoglobin (sus scrofa), myoglobin (equine), myoglobin (human), and myoglobin (sperm whale). Among them, myoglobin (sperm whale) and myoglobin (sus scrofa) are preferable. Examples of the peroxidase include horseradish peroxidase, chloroperoxidase, and catalase. Among them, horseradish peroxidase is preferable.

  In the present invention, the formula (II)

The hemoprotein variant represented by is one in which one amino acid residue is replaced with a cysteine residue in the amino acid sequence of the heme protein. For example, in the case of cytochrome, such a hemoprotein variant is (a) a protein comprising an amino acid sequence in which one amino acid of the amino acid sequence represented by SEQ ID NO: 1 is substituted with cysteine, and (b) SEQ ID NO: 1. It means a protein that consists of an amino acid sequence in which one or two amino acids are deleted, substituted, or added in the amino acid sequence in which one amino acid in the amino acid sequence represented is substituted with cysteine, and functions as cytochrome. The amino acid sequence represented by SEQ ID NO: 1 is the amino acid sequence of natural cytochrome b ₅₆₂ . The amino acid sequence of natural cytochrome b ₅₆₂ is not limited to the amino acid sequence represented by SEQ ID NO: 1, and an appropriate one may be selected in the Protein Data Bank. Specific examples include ID number 1QPU (June 2, 1999), and the amino acid sequence represented by SEQ ID NO: 1 corresponds to that of ID number 1QPU. The one amino acid residue that is replaced by cysteine is preferably an amino acid residue that is located outside the structure when the cytochrome takes a three-dimensional structure. Moreover, it is necessary that the replacement with a cysteine residue does not affect the structure of the heme contained in the cytochrome. In order to perform such substitution, for example, in the amino acid sequence of SEQ ID NO: 1, the first to 106th amino acid residues, preferably 60, 62, 63, 66, 67, 70, 73, 74, 76, 78, The 89th, 90th, 96th, 99th and 100th amino acid residues, more preferably the 60th, 63rd, 66th, 67th and 100th amino acid residues can be replaced with cysteine residues.

  In addition, for example, in the case of hemoglobin, such a hemoprotein variant is (a) a protein comprising an amino acid sequence in which one amino acid of the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 5 is substituted with cysteine, ( b) an amino acid sequence in which one or two amino acids are deleted, substituted or added in the amino acid sequence in which one amino acid of the amino acid sequence represented by SEQ ID NO: 4 or 5 is substituted with cysteine, and hemoglobin A protein that functions as (human). The amino acid sequence represented by SEQ ID NO: 4 is the amino acid sequence of the natural hemoglobin α subunit, and the amino acid sequence represented by SEQ ID NO: 5 is the amino acid sequence of the β subunit of natural hemoglobin. The amino acid sequence of natural hemoglobin is not limited to the amino acid sequences represented by SEQ ID NO: 4 and SEQ ID NO: 5, and an appropriate one may be selected in Protein Data Bank. Specific examples include those with ID number 1GZX (July 8, 2002), and the amino acid sequences represented by SEQ ID NO: 4 and SEQ ID NO: 5 correspond to those with ID number 1GZX. The one amino acid residue that can be replaced with cysteine is preferably an amino acid residue that is located outside the structure when the hemoglobin takes a three-dimensional structure. Moreover, it is necessary that the replacement with a cysteine residue does not affect the structure of the heme contained in the hemoglobin.

  For example, in the case of myoglobin, such a variant of heme protein is (a) a protein comprising an amino acid sequence in which one amino acid of the amino acid sequence represented by SEQ ID NO: 2 is substituted with cysteine, and (b) SEQ ID NO: It means an amino acid sequence in which one or two amino acids are deleted, substituted or added in the amino acid sequence in which one amino acid of the amino acid sequence represented by 2 is substituted with cysteine, and which functions as myoglobin. The amino acid sequence represented by SEQ ID NO: 2 is an amino acid sequence of wild-type myoglobin (sperm whale). The amino acid sequence represented by SEQ ID NO: 2 further includes Met as an amino acid residue before the first amino acid residue Val of the natural amino acid sequence (the amino acid sequence of the protein actually extracted from whale). . Furthermore, aspartic acid, which is the 122nd amino acid residue in the natural amino acid sequence, corresponds to the 123rd amino acid sequence represented by SEQ ID NO: 2, but the amino acid residue is replaced by asparagine. Yes. The amino acid sequence of wild-type myoglobin is not limited to the amino acid sequence represented by SEQ ID NO: 2, and an appropriate one may be selected in the Protein Data Bank. Specific examples include those with ID number 2 MBW (June 20, 1996), and the amino acid sequence represented by SEQ ID NO: 2 corresponds to that with ID number 2 MBW. The one amino acid residue that can be replaced by cysteine is preferably an amino acid residue that is located outside the structure when the myoglobin takes a three-dimensional structure. Moreover, it is necessary that the replacement with a cysteine residue does not affect the structure of the heme contained in the myoglobin. In order to perform such substitution, for example, in the amino acid sequence of SEQ ID NO: 2, the first to 154th amino acid residues, preferably the ninth, 13, 17, 54, 103, 107, 114, 118, 122, 126, The 127th, 134th, 141st and 148th amino acid residues, more preferably the 126th and 134th amino acid residues can be replaced with cysteine residues.

  In addition, for example, in the case of horseradish peroxidase, such a variant of heme protein is (a) a protein comprising an amino acid sequence in which one amino acid of the amino acid sequence represented by SEQ ID NO: 3 is substituted with cysteine, (b) A protein comprising an amino acid sequence in which one or two amino acids are deleted, substituted or added in the amino acid sequence in which one amino acid of the amino acid sequence represented by SEQ ID NO: 1 is substituted with cysteine, and having horseradish peroxidase activity Means. The amino acid sequence represented by SEQ ID NO: 3 is the amino acid sequence of natural horseradish peroxidase. The amino acid sequence of natural horseradish peroxidase is not limited to that represented by SEQ ID NO: 3, and an appropriate one may be selected in Protein Data Bank. Specific examples include those with ID number 1ATJ (February 4, 1998), and the amino acid sequence represented by SEQ ID NO: 3 corresponds to that with ID number 1ATJ. The one amino acid residue to be replaced with cysteine is preferably an amino acid residue that is located outside the structure when the horseradish peroxidase takes a three-dimensional structure. Moreover, it is necessary that the replacement with a cysteine residue does not affect the structure of heme contained in the horseradish peroxidase.

  In the mutant, as described above, -SH means the side chain thiol group of the cysteine residue, the Heme means natural heme, and the formula HS-X shows the apo form of the mutant. means.

In the structure of the present invention, in the formula (IA) or (IB), R ¹ , R ² , R ³ and R ⁴ each independently represents a lower alkyl group or a lower alkenyl group,
Y is a group represented by -Z1-Y1-Z2-Y2- or -Z1-Y1-, wherein Z1 is a group bonded to -CO- in formula (IA) and formula (IB). Means
Z1 is selected from the group consisting of O, NH and S;
Y1 and Y2 are each independently formula _{- (CH 2) n1 -,} - (C 6 H 4) n5 -, - (CH 2) n1 - (C 6 H 4) n5 -, - (C 6 H _{4) n5 - (CH 2)} n1 -, - (CH 2) n1 - (C 6 H 4) n5 - (CH 2) n2 -, - (CH 2) n1 -O- (CH 2) n2 -, - _{(CH 2) n1 -O- (CH} 2) n2 -O- (CH 2) n3 - or _{- (CH 2) n1 -O- (} CH 2) n2 -O- (CH 2) n3 -O- (CH ₂ ) a group represented by _n4- , wherein n1, n2, n3, n4 and n5 each independently represents an integer of 1 to 20,
Z2 is selected from the group consisting of NHCO, CONH, NH, O, N = N, N = CH, CH = N, C = C;
A structure in which M is Fe ²⁺ , Fe ³⁺ , Zn ²⁺ Co ²⁺ , Co ³⁺ , Mn ^2+, or Mn ³⁺ or a protein polymer represented by the formula (IA) or (IB) or a salt thereof is cross-linked.

In the structure of the present invention, in the formula (IA), R ¹ , R ² , R ³ and R ⁴ each independently represents a lower alkyl group or a lower alkenyl group,
Y is a group represented by -Z1-Y1-Z2-Y2- or -Z1-Y1-, wherein Z1 is a group bonded to -CO- in formula (IA) and formula (IB). Means
Z1 is selected from the group consisting of O, NH and S;
Y1 and Y2 are each independently formula _{- (CH 2) n1 -,} - (C 6 H 4) n5 -, - (CH 2) n1 - (C 6 H 4) n5 -, - (C 6 H _{4) n5 - (CH 2)} n1 -, - (CH 2) n1 - (C 6 H 4) n5 - (CH 2) n2 -, - (CH 2) n1 -O- (CH 2) n2 -, - _{(CH 2) n1 -O- (CH} 2) n2 -O- (CH 2) n3 - or _{- (CH 2) n1 -O- (} CH 2) n2 -O- (CH 2) n3 -O- (CH ₂ ) a group represented by _n4- , wherein n1, n2, n3, n4 and n5 each independently represents an integer of 1 to 20,
Z2 is selected from the group consisting of NHCO, CONH, NH, O, N = N, N = CH, CH = N, C = C;
A structure in which M is Fe ²⁺ , Fe ³⁺ , Zn ²⁺ Co ²⁺ , Co ³⁺ , Mn ^2+, or Mn ³⁺ or a structure in which a protein polymer represented by the formula (IA) or a salt thereof is cross-linked is preferable.

In the structure of the present invention, in the formula (IA), R ¹ , R ² , R ³ and R ⁴ each independently represents a lower alkyl group or a lower alkenyl group,
Y is a group represented by -Z1-Y1-Z2-Y2- or -Z1-Y1-, wherein Z1 is a group bonded to -CO- in formula (IA) and formula (IB). Means
Z1 is selected from the group consisting of O, NH and S;
Y1 and Y2 are each independently formula _{- (CH 2) n1 -,} - (C 6 H 4) n5 -, - (CH 2) n1 - (C 6 H 4) n5 -, - (C 6 H _{4) n5 - (CH 2)} n1 -, - (CH 2) n1 - (C 6 H 4) n5 - (CH 2) n2 -, - (CH 2) n1 -O- (CH 2) n2 -, - _{(CH 2) n1 -O- (CH} 2) n2 -O- (CH 2) n3 - or _{- (CH 2) n1 -O- (} CH 2) n2 -O- (CH 2) n3 -O- (CH ₂ ) a group represented by _n4- , wherein n1, n2, n3, n4 and n5 each independently represents an integer of 1 to 20,
Z2 is selected from the group consisting of NHCO, CONH, NH, O, N = N, N = CH, CH = N, C = C;
A structure in which a protein polymer represented by the formula (IA) or a salt thereof is cross-linked, wherein M is Fe ²⁺ or Fe ³⁺ is preferable.

In the structure of the present invention, in the formula (IA), R ¹ and R ³ are each independently a lower alkyl group, and R ² and R ⁴ are each independently a lower alkenyl group. And
Y is a group represented by -Z1-Y1-Z2-Y2- or -Z1-Y1-, wherein Z1 represents a group bonded to -CO- in formula (IA);
Z1 is selected from the group consisting of O, NH and S;
Y1 and Y2 are each independently formula _{- (CH 2) n1 -,} - (C 6 H 4) n5 -, - (CH 2) n1 - (C 6 H 4) n5 -, - (C 6 H _{4) n5 - (CH 2)} n1 -, - (CH 2) n1 - (C 6 H 4) n5 - (CH 2) n2 -, - (CH 2) n1 -O- (CH 2) n2 -, - _{(CH 2) n1 -O- (CH} 2) n2 -O- (CH 2) n3 - or _{- (CH 2) n1 -O- (} CH 2) n2 -O- (CH 2) n3 -O- (CH ₂ ) a group represented by _n4- , wherein n1, n2, n3, n4 and n5 each independently represents an integer of 1 to 20,
Z2 is selected from the group consisting of NHCO, CONH, NH, O, N = N, N = CH, CH = N, C = C;
M is Fe ²⁺ , Fe ³⁺ , Zn ²⁺ Co ²⁺ , Co ³⁺ , Mn ²⁺ or Mn ³⁺
A structure in which the protein polymer represented by the formula (IA) or a salt thereof is cross-linked is more preferable, wherein the heme protein is cytochrome or myoglobin.

In the structure of the present invention, in the formula (IA), R ¹ and R ³ are each independently a lower alkyl group, and R ² and R ⁴ are each independently a lower alkenyl group. And
Y is --Z1-Y1-, Z1 is O or NH, Y1 is the formula _{- (CH 2) n1 -,} - (CH 2) n1 -O- (CH 2) n2 -O- ( CH _{2) n3} - or _{- (CH 2) n1 -O- (} CH 2) n2 -O- (CH 2) n3 -O- (CH 2) n4 - a group represented by where n1, n2, n3 and n4 each independently represents an integer of 1 to 3, M is Fe ²⁺ , Fe ³⁺ , Zn ²⁺ , Co ²⁺ , Co ³⁺ , Mn ²⁺ or Mn ³⁺ , and the heme protein is cytochrome or myoglobin A structure in which a protein polymer represented by the formula (IA) or a salt thereof is crosslinked is more preferable.

In the structure of the present invention, in the formula (IA), R ¹ and R ³ are each independently a methyl group, R ² and R ⁴ are each independently a vinyl group, Y is --Z1-Y1-, Z1 is NH, Y1 is the formula _{- (CH 2) 2 -,} - (CH 2) 2 -O- (CH 2) 2 -O- (CH 2 ) ₂ — or — (CH ₂ ) ₃ —O— (CH ₂ ) ₂ —O— (CH ₂ ) ₂ —O— (CH ₂ ) ₃ —, wherein M is Fe ²⁺ , Fe ³⁺ _, Zn ²⁺ , Co ²⁺ , Co ³⁺ , Mn ²⁺ or Mn ³⁺ , the heme protein is cytochrome having the amino acid sequence represented by SEQ ID NO: 1, and the one amino acid residue is the 63rd histidine Or the amino acid represented by SEQ ID NO: 2 A structure in which the protein polymer represented by the formula (IA) or a salt thereof is cross-linked is myoglobin having a sequence and the one amino acid residue is the 126th alanine.

In the structure of the present invention, in Formula (IA), R ¹ and R ³ are each independently a methyl group, and R ² and R ⁴ are each independently a vinyl group. and, Y is a --Z1-Y1-, Z1 is NH, Y1 is the formula _{- (CH 2) 2 -,} - (CH 2) 2 -O- (CH 2) 2 -O- ( CH ₂ ) ₂ — or — (CH ₂ ) ₃ —O— (CH ₂ ) ₂ —O— (CH ₂ ) ₂ —O— (CH ₂ ) ₃ —, and M is Fe ²⁺ or Fe ³⁺ , the hemoprotein is cytochrome having the amino acid sequence represented by SEQ ID NO: 1, and the one amino acid residue is the 63rd histidine, or the amino acid sequence represented by SEQ ID NO: 2 A myoglobin having the one amino acid Group protein polymer or a salt thereof represented by formula (IA) is the 126th alanine has been crosslinked structure is more preferred.

  In the structure of the present invention, the protein polymer salt is a protein polymer salt represented by the formulas (IA) and (IB), for example, an alkali metal salt such as sodium or potassium, or an alkaline earth metal such as calcium or magnesium. Salts, salts with inorganic bases such as ammonium salts, and organic amine salts such as triethylamine, pyridine, picoline, ethanolamine, triethanolamine, dicyclohexylamine, N, N′-dibenzylethylenediamine, and hydrochloric acid, hydrobromic acid , Inorganic acid salts such as sulfuric acid and phosphoric acid, and organic carboxylic acid salts such as formic acid, acetic acid, trifluoroacetic acid, maleic acid and tartaric acid, and sulfonic acid addition such as methanesulfonic acid, benzenesulfonic acid and p-toluenesulfonic acid Salt, and arginine, aspartic acid, gluta Salts or acid addition salts with bases such as basic or acidic amino acids such as phosphate and the like.

  Next, an example of the manufacturing method of the structure of this invention by which protein polymer or its salt was bridge | crosslinked is demonstrated.

  In the production method, the protein polymer represented by the general formula (IA) or (IB) or a salt thereof is subjected to a condition for forming a covalent bond between molecules, and represented by the general formula (IA) or (IB). A protein polymer or a salt thereof is crosslinked between molecules to obtain the structure. The conditions for forming a covalent bond between the molecules are not particularly limited, but are, for example, conditions for generating radicals. Examples of the conditions for generating the radical include contact with peracid such as hydrogen peroxide, peracetic acid, metachloroperbenzoic acid, cumene hydroperoxide, X-ray irradiation, electron beam irradiation, ion beam irradiation, gamma ray irradiation, light (UV) irradiation is mentioned, and contact with hydrogen peroxide is preferable.

  In the production method described above, when the condition for forming a covalent bond between molecules is contact with hydrogen peroxide, the protein polymer or a salt thereof is added with hydrogen peroxide to the metalloporphyrin contained therein, for example, 0.01 to 100 equivalents, preferably 0.1 to 5 equivalents, more preferably 0.5 to 1.5 equivalents. At this time, the protein polymer or a salt thereof can be reacted with hydrogen peroxide in an aqueous solution. The concentration of the protein polymer or a salt thereof in the aqueous solution is, for example, 0.01 to 5 mM, preferably 0.1 to 1 mM. The contact between the protein polymer or a salt thereof and hydrogen peroxide can be performed, for example, in the range of 2 to 60 ° C, preferably in the range of 20 to 30 ° C. The contact between the protein polymer or a salt thereof and hydrogen peroxide can be performed, for example, for 0.1 to 72 hours, preferably 2 to 24 hours.

  Alternatively, the condition for forming the covalent bond is preferably a condition for reacting with a crosslinking agent having a functional group capable of binding to side chains of two amino acids among the amino acids constituting the protein polymer. Examples of the functional group that reacts with the crosslinking agent include formyl group, imidoester group, N-hydroxysuccinimide ester group, isocyanate group (the side chain of the corresponding amino acid is an amino group), methanethiosulfonate group , Maleimide group, iodoacetamide group (the side chain of the corresponding amino acid is —SH group), diazo group (the side chain of the corresponding amino acid is carboxyl group) and the like. Examples of the cross-linking agent include a homo-bifunctional linker and a hetero-bifunctional linker. Examples of the homobifunctional linker include glutaraldehyde, malondialdehyde, adipaldehyde, dimethyl adipimidate, dimethyl suberimidate, dimethyl pimelimate, bis (N-hydroxysuccinimide ester), bis (sulfosuccinimidyl). ) Suberate, 3,3′-dithiodipropionic acid di (N-succinimidyl), 3,3′-dithiobis (sulfosuccinimidyl sulfonate), 1,6-hexamethylene diisocyanate, S, S′- Hexamethylene bis (methanethiosulfonate), N, N′-trimethylene bismaleimide, N, N′-ethylene bis (2-iodoacetamide), 1,6-diazohexane) and the like. Examples of the heterobifunctional linker include N-succinimidyl 3- (2-pyridyldithio) propionate, succinimidyl 4 (N-maleimidomethyl) cyclohexane-1-carboxylate, and pyridyl-2,2′-dithiobenzyl. Examples include diazoacetate.

  In the production method, when the condition for forming a covalent bond between molecules is a condition for reacting with the cross-linking agent, the reaction condition for the protein polymer or a salt thereof and the cross-linking agent is within the scope of common technical knowledge in the field. If so, there is no particular limitation.

  The protein polymer represented by the general formula (IA) or (IB) used in the production method of the present invention can be produced, for example, according to JP-A-2008-22565.

  The protein polymer represented by the formula (IA) is obtained, for example, as follows (see Scheme 1).

  The protein polymer represented by the formula (IB) can be obtained, for example, as follows (see Scheme 2).

Formula (IA), (IB), (III-1A), (III-2A), (III-1B), (III-2B), (IV-1A), (IV-2A), (IV-1B) ), (IV-2B), (V-1A), (V-2A), (V-1B) and formula (V-2B),
R ¹ , R ² , R ³ and R ⁴ each independently represents a hydrogen atom, a lower alkyl group, a lower alkyl group or a lower alkenyl group substituted with a halogen atom,
Y is a group represented by -Z1-Y1-Z2-Y2- or -Z1-Y1-, wherein Z1 is a group bonded to -CO- in formula (IA) and formula (IB). Means
Z1 is selected from the group consisting of O, NH and S;
Y1 and Y2 are each independently formula _{- (CH 2) n1 -,} - (C 6 H 4) n5 -, - (CH 2) n1 - (C 6 H 4) n5 -, - (C 6 H _{4) n5 - (CH 2)} n1, - (CH 2) n1 - (C 6 H 4) n5 - (CH 2) n2 -, - (CH 2) n1 -O- (CH 2) n2 -, - ( _{CH 2) n1 -O- (CH 2} ) n2 -O- (CH 2) n3 - or _{- (CH 2) n1 -O- (} CH 2) n2 -O- (CH 2) n3 -O- (CH 2 A group represented by _n4- , wherein n1, n2, n3, n4 and n5 each independently represents an integer of 1 to 20,
Z2 is selected from the group consisting of NHCO, CONH, NH, O, N = N, N = CH, CH = N, C = C;
M is selected from the group consisting of Fe ²⁺ , Fe ³⁺ , Zn ²⁺ , Co ²⁺ , Co ³⁺ , Mn ²⁺ and Mn ³⁺ ,
m and n are each an integer, 1 ≦ m + n ≦ 1000,
X means X in the hemoprotein mutant, and the hemoprotein mutant is represented by the formula (II).

  The mutant is obtained by replacing one amino acid residue with a cysteine residue in the amino acid sequence of a natural heme protein. In the mutant, -SH means a side chain thiol group of the cysteine residue, Heme means natural heme, and formula HS-X means an apo form of the mutant.

  In the formula, the definitions of the lower alkyl group and the lower alkenyl group are as described above.

  In the production method, reaction of a metalloporphyrin linker represented by formula (IV-1A), (IV-2A), (IV-1B) or formula (IV-2B) with a heme protein represented by formula (II) Can be carried out in a solvent in the presence of a catalyst under an inert gas (eg, argon, nitrogen, etc.) atmosphere. Examples of the catalyst include weak acids and weak bases. Examples of the weak acid include citric acid, hydrochloric acid, sulfuric acid, nitric acid, p-toluenesulfonic acid, and acetic acid. Examples of the weak base include histidine, sodium hydrogen carbonate, tris (hydroxymethyl) aminomethane and the like.

  In the above production method, the reaction solvent is not limited. For example, alcohols (for example, methanol, ethanol, etc.), polar solvents such as water, dimethyl sulfoxide, dimethylformamide, buffer solutions (for example, Tris-HCl buffer solution, phosphate buffer). Liquid, borate buffer, carbonate buffer) and the like. The reaction solvent is represented by a metal porphyrin linker represented by formula (IV-1A), (IV-2A), (IV-1B) or formula (IV-2B) and / or general formula (II). You may contain the additive for improving the solubility of heme protein. Examples of the additive include histidine. The said solvent may use 1 type, or may mix and use 2 or more types. The solvent is preferably selected from the dimethyl sulfoxide and buffer that may be added as described above, and more preferably a mixture of dimethyl sulfoxide and Tris-HCl buffer, water containing histidine, and the like.

  In the said manufacturing method, although reaction temperature is not limited, For example, it is 0 to 100 degreeC, Preferably it is 10 to 60 degreeC, More preferably, it is 20 to 30 degreeC.

  In the said manufacturing method, although reaction time is not limited, For example, it is 30 minutes-24 hours, Preferably it is 2 hours-10 hours, More preferably, it is 5 hours-8 hours.

  Heme represented by the formula (II) used for the metalloporphyrin linker represented by the formula (IV-1A), (IV-2A), (IV-1B) or the formula (IV-2B) in the production method The molar ratio of protein (metal porphyrin linker represented by formula (IV-1A), (IV-2A), (IV-1B) or formula (IV-2B): heme protein represented by formula (II)) is: For example, 100: 1 to 5: 1, preferably 50: 1 to 10: 1, more preferably 20: 1 to 10: 1.

  In the production method, the metalloporphyrin linker represented by the formula (IV-1A), (IV-2A), (IV-1B) or formula (IV-2B) may be obtained commercially or publicly known. You may make your own by referring to the literature.

  In the production method, the heme protein represented by the formula (II) can be produced, for example, as follows.

  The gene of the heme protein represented by the above formula (II) may be cloned using guinea pig total RNA or the like based on the base sequence of the natural heme protein gene, or using the phosphoramidite method. Then, the DNA may be chemically synthesized. The cloning method is not particularly limited, and can be performed using, for example, a commercially available cloning kit. In addition, the gene of the heme protein represented by the formula (II) may be introduced into the host cell.

  Examples of the host cell include animal cells, plant cells, insect cells, yeasts, bacteria, and the like. Examples of the gene introduction method include a lithium acetate method, a calcium phosphate method, a method using a liposome, electroporation, a method using a viral vector, a micropipette injection method, and the like. Alternatively, it may be introduced using an integration type into a host chromosome or an artificial chromosome or plasmid type capable of autonomous replication / distribution.

  The gene to be introduced into the host cell is preferably operably linked to necessary regulatory sequences so that it is constitutively or arbitrarily expressed in the host cell. The regulatory sequence is a base sequence necessary for expression of the gene operably linked in a host cell. For example, examples of regulatory sequences suitable for eukaryotic cells include promoters, polyadenylation signals, enhancers. Etc. Said operatively connected means that the components are juxtaposed so that they can perform their functions.

  Moreover, an example of the manufacturing method of the protein monomer represented by the said Formula (V-1A), (V-2A), (V-1B) or Formula (V-2B) or its salt is demonstrated.

  The production method is carried out by treating a protein monomer represented by the formula (III-1A), (III-2A), (III-2A) or the formula (III-2B) or a salt thereof with an acid (V- A protein monomer represented by 1A), (V-2A), (V-1B) or formula (V-2B) or a salt thereof is obtained.

  In the production method, the acid treatment reaction of the protein monomer represented by the formula (III-1A), (III-2A), (III-2A) or the formula (III-2B) or a salt thereof is, for example, hydrochloric acid, sulfuric acid, It is performed by treating with nitric acid or the like.

  In the above production method, the reaction solvent is not limited. For example, alcohols (for example, methanol, ethanol, etc.), polar solvents such as water, dimethyl sulfoxide, dimethylformamide, buffer solutions (for example, Tris-HCl buffer solution, phosphate buffer). Liquid, borate buffer, carbonate buffer) and the like. The said solvent may use 1 type, or may mix and use 2 or more types. The solvent is preferably selected from dimethyl sulfoxide and a buffer solution, and more preferably a mixture of dimethyl sulfoxide and a tris hydrochloric acid buffer solution, a phosphate buffer solution, or the like.

  In the said manufacturing method, although reaction temperature is not limited, For example, it is 0 to 30 degreeC, Preferably it is 0 to 10 degreeC, More preferably, it is 3 to 5 degreeC.

  In the said manufacturing method, acid treatment can be performed by pH 0.5-3.0, for example, Preferably it is 1.5-2.5, More preferably, it is 1.8-2.1.

  The protein monomer represented by the formula (III-1A), (III-2A), (III-2A) or the formula (III-2B) or a salt thereof, and the formula (V-1A), (V-2A), ( In the method for producing a protein monomer represented by V-1B) or formula (V-2B) or a salt thereof, the hemoprotein is preferably cytochrome, hemoglobin, myoglobin, or peroxidase.

  Next, an example of a method for producing a protein polymer represented by the general formula (IA) or (IB) or a salt thereof will be described.

  The production method comprises treating the protein monomer represented by the formula (V-1A), (V-2A), (V-1B) or the formula (V-2B) or a salt thereof under neutral conditions, A protein polymer or a salt thereof is obtained.

  In the above production method, the reaction solvent is not limited. For example, alcohols (for example, methanol, ethanol, etc.), polar solvents such as water, dimethyl sulfoxide, dimethylformamide, buffer solutions (for example, Tris-HCl buffer solution, phosphate buffer). Liquid, borate buffer, carbonate buffer) and the like. The said solvent may use 1 type, or may mix and use 2 or more types. The solvent is preferably selected from buffer solutions, more preferably Tris-HCl buffer solution, phosphate buffer solution and the like.

  In the said manufacturing method, although reaction temperature is not limited, For example, it is 0 to 30 degreeC, Preferably it is 0 to 10 degreeC, More preferably, it is 3 to 5 degreeC.

  In the production method, the treatment under neutral conditions may be performed at a pH of, for example, 6.0 to 10.0, preferably 6.5 to 8.5, more preferably 7.0 to 8.0. it can.

  In the production method, the protein monomer represented by the formula (V-1A) or a salt thereof is in a positional isomer relationship with the protein monomer represented by the formula (V-2A) or a salt thereof. In addition, the protein monomer represented by the formula (V-1B) or a salt thereof is in a positional isomer relationship with the protein monomer represented by the formula (V-2B) or a salt thereof. When the reaction proceeds while mixing at the stage of the metal porphyrin linker represented by the formula (IV-1A) and the metal porphyrin linker represented by the formula (IV-2A), the resulting protein monomer has the formula (V-1A ) And a protein monomer represented by the formula (V-2A). In that case, the finally obtained protein polymer is a copolymer represented by the formula (IA). The same applies to Formula (V-1B) or Formula (V-2B).

  The structure in which the protein polymer of the present invention or a salt thereof is crosslinked is useful as a biopolymer that functions as an electron pool when the heme protein is cytochrome. When the hemoprotein is hemoglobin or myoglobin, it is useful as a biopolymer for storing or transporting oxygen. When the heme protein is peroxidase, it is useful as an assembly of enzymes.

  The structure in which the protein polymer of the present invention or a salt thereof is crosslinked is represented by the formula (V-1A), (V-2A), (V-1B) or the formula (V-2B) under acidic or basic conditions. A protein monomer represented by the formula (V-1A), (V-2A), (V-1B) or a dimer of the salt represented by the formula (V-2B), or a salt thereof; V-1A), (V-2A), (V-1B) or a protein monomer represented by the formula (V-2B) or a salt thereof can be decomposed. Therefore, the structure in which the protein polymer of the present invention or a salt thereof is cross-linked is useful as a pH-responsive protein polymer that controls the aggregation state by pH.

  The structure in which the protein polymer of the present invention or a salt thereof is cross-linked can change its structure according to pH as described above. For example, the structure of the present invention can be used as a drug delivery material by conjugating a drug to the structure of the present invention.

  The present invention will be described more specifically with reference to the following examples. However, the scope of the present invention is not limited by the following examples.

  Various spectra were measured using the following instrument.

Nuclear magnetic resonance (NMR) spectrum was measured using JEOL EX270 nuclear magnetic resonance apparatus (270 MHz) or Bruker DPX-400 nuclear magnetic resonance apparatus (400 MHz), and the residual signal of the measurement solvent was used as an internal reference. Electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) was measured using an Applied Biosystems Mariner API-TOF workstation (Workstation). The ultraviolet-visible absorption spectrum was measured using a self-recording spectrophotometer UV-2550 or UV-3150 manufactured by Shimadzu Corporation. The pH of the aqueous solution was measured using a pH meter F-52 manufactured by Horiba. Size exclusion chromatography (SEC) is based on the GE Healthcare AKTA _FPLC system with Superdex 200 10/300 GL column (exclusion limit 1.3 × 10 ⁶ Da) or Shodex KW405-4F column (discharge limit). 2 × 10 ⁷ Da) was connected and the detection was performed using a UPC-900 detector. The atomic force microscope (AFM) was measured using a Nanoscope V manufactured by Digital Instruments. For electrophoresis measurement, measurement was performed using a fully automated electrophoresis system Phassystem manufactured by GE Healthcare. The scanning electron microscope (SEM) was measured using S-5500 manufactured by Hitachi High-Tech.

The starting material was produced with reference to the following documents.
Protoporphyrin IX mono t-butyl ester 2: T. Matsuo, T. Hayashi, Y. Hisaeda, J. Am. Chem. Soc. 124, 11234 (2002).
Mono N-Boc protected diamines: M. Trester-Zedlitz, K. Kamada, SK Burley, D. Fenyo, BT Chait, TW Muir, J. Am. Chem. Soc. 125, 2416 (2003); and R. Schneider F. Schmitt, C. Frochot, Y. Fort, N. Lourette, F. Guillemin, J.-F. Mueller, M. Barberi-Heyob, Bioorg. Med. Chem. 13, 2799 (2005).
N-methoxycarbonylmaleimide: O. Keller, J. Rudinger, Helv. Chim. Acta. 58, 531 (1975).
As other general reagents, commercially available products were used as they were.

(1) Preparation of Myoglobin Mutant (A125C) Dimer For Escherichia coli having a mutant protein plasmid, literature formulation (S. Hirota, K. Azuma, M. Fukuda, S. Kuroiwa, N. Funasaki, Biochemistry 44, 10322 (2005)). The mutant protein is Escherichia coli strain TB-1 and abundant in the same manner as wild-type myoglobin expression according to the literature prescription (BA Springer, SG Sliger, Proc. Natl. Acad. Sci. USA 84, 8961 (1987)). Expressed. The expressed proteins were anion exchange column (DEAE Sepharose FF, 2.7 cm × 10 cm), cation exchange column (CM-52, 2.7 cm × 18 cm) and gel filtration column (Sephadex G-50, 1.5 cm × 100 cm). The fraction of myoglobin mutant A125C dimer derived from sperm whale was recovered and used in the following experiments.

(2) Production of Compound represented by Formula 1 (13) The compound represented by Formula 1 (13) was produced according to Scheme 3 below.

  In scheme 3, each “positional isomer” is as follows. The positional isomer of compound 2 is compound 2 ′, the positional isomer of compound 3 (13) is compound 3 (13) ′, the positional isomer of compound 4 (13) is compound 4 (13) ′, and compound 5 (13). The positional isomer of is compound 5 (13) ′, and the positional isomer of compound 1 (13) is compound 1 (13) ′.

(I) Production of Compound Represented by Formula 3 (13) and its Regioisomer In a 50 mL eggplant-shaped flask under a nitrogen atmosphere, protoporphyrin IX mono t-butyl ester 2 and its regioisomer (100 mg, 1.6 × 10 ⁻⁴ mol), N-Boc-bis [2- (3-aminopropoxy) ethyl] ether (diamine (13), 155 mg, 4.9 × 10 ⁻⁴ mol) and DMF (25 mL) are added and dissolved. It was. The solution was cooled by an ice bath, into which a DMF solution (1 mL) of diphenyl phosphate azide (DPPA, 111 mg, 4.0 × 10 ⁻⁴ mol) and triethylamine (Et ₃ N, 65 mg, 4.5 × 10 ^−4). mol) in DMF (1 mL) was added. The solution was stirred at room temperature for 4 hours in the dark, and the same amount of DPPA and Et ₃ N in DMF were added again. After further stirring for 2 hours, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel chromatography (chloroform / acetone = 5/1). The resulting solid was dissolved in a minimum amount of chloroform, and the precipitate formed by adding hexane thereto was collected to give a mixture of the title compound 3 (13) and its regioisomer (73.3 mg, 50%). , Purple, solid)).

¹ H-NMR (270 MHz, pyridine-d ₅ ) δ 10.34 (s, 1H) 10.21 (s, 0.5H) 10.17 (s, 1H) 10.14 (s, 1H) 10.03 (s, 0.5H) 9.95 (s, 0.5H) 8.39-8.25 (m, 2H) 6.41-6.14 (m, 4H) 4.60-4.38 (m, 4H) 3.62-3.45 (m, 16H) 3.34-3.15 (m, 12H) 3.06 (m, 2H) 2.76 (m, 2H) 2.61 (m, 2H) 2.44 (m, 2H) 1.74 (m, 2H) 1.49 (s, 9H) 1.43 (s, 9H) 1.39 (m, 2H), −3.80 (s, 2H);
ESI-TOF-MS (positive mode) m / z 921.83 (M + H) +, calculated for C ₅₃ H ₇₂ N ₆ O ₈ : 922.18;
UV-vis (CHCl ₃ ) λmax / nm (absorbance) 630 (0.032) 576 (0.042) 540 (0.072) 506 (0.088) 408 (1.05).

(Ii) Production of compound represented by formula 4 (13) In a nitrogen atmosphere, compound 3 (13) and its positional isomer (63.3 mg, 6.9 × 10 ⁻⁵ mol), dichloromethane ( 5 mL) and trifluoroacetic acid (TFA, 2 mL) were added with cooling in an ice bath, respectively. Thereafter, the reaction solution was returned to room temperature and stirred. After 7 hours, the solvent was distilled off under reduced pressure, and a minimum amount of methanol was added to the resulting residue to dissolve it. Diethyl ether was added thereto, and the resulting precipitate was collected to obtain a mixture of the title compound 4 (13) and its regioisomer (43 mg, 82%, purple, solid).

¹ H NMR (270 MHz, pyridine-d ₅ ) δ 10.41 (s, 1H) 10.19 (s, 0.5H) 10.14 (br, 1.5H) 10.00 (s, 0.5H) 9.94 (s, 0.5H) 8.42-8.27 (m, 2H) 6.43-6.12 (m, 4H), 4.60-4.41 (m, 4H), 3.61-3.47 (m, 16H) 3.33-3.22 (m, 8H) 3.13 (m, 2H) 2.93 (m, 2H ) 2.80 (m, 2H) 2.71 (m, 2H) 1.90 (m, 2H) 1.44 (m, 2H) -3.88 (s, 2H);
ESI-TOF-MS (positive mode) m / z 765.55 (M-TFA) +, calculated for C ₄₄ H ₅₇ N ₆ O ₆ : 765.96;
UV-vis (MeOH) λmax / nm (absorbance) 628 (0.035) 574 (0.044) 538 (0.073) 504 (0.090) 401 (1.02).

(Iii) Production of compound represented by formula 5 (13) In a 50 mL eggplant-shaped flask, compound 4 (13) and its positional isomer (35.0 mg, 4.6 × 10 ⁻⁵ mol), iron (II) chloride Hydrate (180 mg) and sodium hydrogen carbonate (7.7 mg) were added, and the mixture was dissolved in a nitrogen-saturated mixed solvent of chloroform and methanol (chloroform / methanol = 10/1, 10 mL), followed by heating under reflux. After 4 hours, the reaction mixture was cooled to room temperature, and the solvent was distilled off under reduced pressure. The residue was dissolved in a mixed solvent of chloroform and methanol (chloroform / methanol = 2/1) and washed with 0.05M hydrochloric acid. The organic layer was separated and dehydrated with anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was dissolved by adding a minimum amount of methanol, and diethyl ether was added thereto, and the resulting precipitate was collected to give a mixture of the title 5 (13) and its regioisomer (38 mg, 92% , Purple, solid).
ESI-TOF-MS (positive mode) m / z 818.70 (M -Cl ^-- HCl) +, calculated for C ₄₄ H ₅₄ FeN ₆ O ₆ : 818.72
UV-vis (MeOH) λmax / nm (absorbance) 601 (0.037) 494 (0.080) 396 (1.07).

(Iv) Production of compound represented by formula 1 (13) In a 100 mL eggplant-shaped flask, compound 5 (13) and its positional isomer (50.0 mg, 5.6 × 10 ⁻⁵ mol) and N-methoxycarbonylmaleimide (120 mg, 7.70 × 10 ⁻⁴ mol) was dissolved in a mixed solvent of acetone and a saturated aqueous solution of sodium bicarbonate (acetone / saturated aqueous solution of sodium bicarbonate = 2/1, 10 mL). After stirring at room temperature for 2 hours, water (40 mL) was added, and the mixture was further stirred for 1 hour. To the mixture, 0.1N HCl aqueous solution and then chloroform were added and extracted with chloroform. The organic layers were combined and dehydrated with anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel chromatography (chloroform / methanol = 5/1). The resulting solid was dissolved in a minimum amount of chloroform and the precipitate formed by adding hexane thereto was collected to give a mixture of the title compound 1 (13) and its regioisomer (23 mg, 46%, dark purple ,solid).

ESI-TOF-MS (positive mode) m / z 898.38 (M−Cl ⁻ ) ⁺ , C ₄₄ H ₄₆ FeN ₆ O ₇ Calculated value: 898.34
UV-vis (CHCl ₃ / MeOH = 1/1, v / v) λmax / nm (absorbance) 596 (0.064) 483 (0.087) 398 (0.78).

  (3) Production of a protein monomer and a protein polymer having the monomer as a monomer unit (see Scheme 4)

  Formula (II-2)

Means a sperm whale-derived myoglobin mutant (A125C), which is a monomer derived from the protein mutant expressed in Example 1 (1), and the mutant is an amino acid sequence (sequence) of wild-type myoglobin. In number 2), the 126th alanine residue is replaced with a cysteine residue. In the mutant, -SH means a side chain thiol group of the cysteine residue, the Heme means natural heme, and the formula HS-X '' means an apo form of the mutant.

  In scheme 4, each “positional isomer” is as follows. The positional isomer of compound 1 (13) is compound 1 (13) ', the positional isomer of compound (III-13) is compound (III-13)', and the positional isomer of compound V-13) is compound (V- 13) ′.

To the stock solution of A125C dimer prepared in Example 1 (1) (200 μL, 1.10 × 10 ⁻³ M (monomer equivalent concentration), 0.1 M phosphate buffer, pH 7.0), Excess DTT (dithiothreitol) was added and dissolved, and then allowed to stand at 2 ° C. for 30 minutes. This solution was purified with a desalting buffer exchange column equilibrated with 0.1 M L-histidine aqueous solution (Hitrap desalting (registered trademark), manufactured by GE Healthcare Bioscience Co., Ltd.), and A125C monomer (II-2) Got. The A125C monomer (II-2) was made up to 1.5 mL with the 0.1 M L-histidine aqueous solution to prepare an A125C monomer solution. Compound 1 (13) (1.1 × 10 ⁻⁶ mol) solution was added to the A125C monomer solution, and the mixture was stirred at room temperature for 24 hours to obtain Compound 1 (13) and its positional isomer. Protein monomer (1II-13) and its positional isomer were obtained by combining A125C monomer (II-2) with A125C. The solution of Compound 1 (13) and its positional isomer was a mixed solvent of 0.1 M L-histidine aqueous solution, DMSO, and dithionite (0.1 M L-histidine aqueous solution: DMSO = 6: 1, dithionite was A125C Compound 1 (13) and its positional isomer (1.1 × 10 ⁻⁶ mol) were dissolved in (equivalent to monomer) (0.6 mL). All the above operations were performed in a glove box under a nitrogen atmosphere.

The reaction solution was transferred to a dialysis membrane (Wako, MWCO 14,000 Da), and dialyzed at 4 ° C. using a phosphate buffer (0.01 M, pH 6.0) (1 L × 1 hour × 2). To a solution of protein monomer (III-13) and its positional isomer obtained by dialysis, 0.1N hydrochloric acid was added to adjust to pH 2.25 to obtain protein monomer (V-13) and its positional isomer. To the solution of the protein monomer (V-13) and its positional isomer, 2-butanone (5 mL × 4) was added for extraction. The aqueous layer was separated, transferred to a dialysis membrane (Wako, MWCO 14,000 Da), and dialyzed at 4 ° C. using a phosphate buffer (0.1 M, pH 7.0) (1 L × 2 hours × 3). . The precipitate was removed from the obtained aqueous solution of protein polymer (I-13) by centrifugation, and the supernatant protein polymer (I-13) solution was concentrated to about 10 ⁻³ M by ultrafiltration and stored in a cool dark place. .

(4) Producing a structure by cross-linking protein polymer Protein polymer (I-13) (heme equivalent concentration 0.3 mM, 10 μL, 100 mM phosphate buffer (pH 7.0)) and hydrogen peroxide aqueous solution (2.25 mM) 2 μL, 100 mM phosphate buffer (pH 7.0)) was added and the mixture was allowed to stand at 25 ° C. for 2 hours to crosslink the protein polymer. The obtained structure was then stored in a cool and dark place.

(5) Producing an oxygen complex of the structure Excess amount of the protein polymer cross-linked structure aqueous solution (0.25 mM (heme equivalent concentration), 30 μL, 0.1 M phosphate buffer, pH 7.0) Dithionite was added and equilibrated with 0.1 M phosphate buffer (pH 7.0). Dithionite was removed from the mixture using HiTrap Desalting (GE Healthcare) to purify the oxygen complex of the structure.

  [Physical properties of the protein polymer obtained in Example 1 and the structure obtained by crosslinking the polymer]

<Manufacture of oxygen complex of protein polymer>
An excess amount of dithionite was added to the aqueous solution (0.25 mM (concentration of heme), 30 μL, 0.1 M phosphate buffer, pH 7.0) of the protein polymer (I-13), and 0.1 M phosphate buffer ( equilibrated at pH 7.0). The mixture was purified by a Sephadex-G25 column to obtain an oxygen complex of the protein polymer.
Further, an oxygen complex of natural myoglobin was obtained in the same manner as described above except that natural myoglobin was used instead of the protein polymer.

(I) Measurement of UV-vis spectrum Fig. 1a shows the UV-vis spectrum of natural myoglobin, and Fig. 1b shows the UV-vis spectrum of the protein polymer (I-13) produced in Example 1. From FIG. 1a and FIG. 1b, it was confirmed that the spectrum of the protein polymer (I-13) had a spectrum very similar to that of the natural myoglobin. Therefore, it was confirmed that the heme of the protein polymer (I-13) in which the porphyrin linker was introduced into the mutant myoglobin was retained in the heme pocket in the same manner as the natural myoglobin.

  Further, FIG. 1c shows the UV-vis spectrum of the oxygen complex of natural myoglobin, and FIG. 1d shows the UV-vis spectrum of the oxygen complex of the protein polymer (I-13). It is known that myoglobin having an oxygen storage capacity forms a very stable oxygen complex by reducing heme iron to divalent with a reducing agent in the atmosphere and exhibits a characteristic ultraviolet-visible absorption spectrum. This FIG. 1c and FIG. 1d have characteristic 417 nm, 543 nm and 580 nm absorption as well. Therefore, it was confirmed that the protein polymer (I-13) having the monomer unit of the protein monomer represented by the formula (V-13) and its positional isomer or salt thereof forms a very stable oxygen complex. . Furthermore, from this, it was confirmed that the protein polymer represented by the formula (I-13) formed a supramolecular assembly while maintaining the original protein function.

(Ii) Measurement of affinity with oxygen The kinetics measurement of the oxygen complex of the protein polymer (I-13) and the natural myoglobin complex was performed at 25 ° C. in a phosphate buffer (100 mM, pH 7.0). . The autooxidation process was measured at 37 ° C. The oxygen binding constant K ^O2 is obtained by dividing the binding rate constant k ^O2 _on by the dissociation rate constant k ^O2 _off . The results obtained from the following measurements are shown in Table 2. The protein polymer (I-13) corresponds to a polymer obtained by polymerizing 11 (n = 11) protein monomers. This protein polymer (I-13) or the entire positional isomer was calculated as one molecule.

[Measurement of oxygen binding rate constant k ^O2 _on ]
Oxygen complex of natural myoglobin or oxygen-binding protein polymer (I-13) in phosphate buffer (100 mM, pH 7.0) in the atmosphere ([O ₂ ] = 2.64 × 10 ⁻⁴ M) Were excited by laser flash photolysis (excitation wavelength: 532 nm, 5 ns pulse). Thereafter, the change in absorbance at 434 nm, which is the maximum absorption wavelength of each of natural myoglobin or the protein polymer produced in Example 1 (Fe (II) heme complex of myoglobin, deoxy-Mb), was followed. The probe light was passed through a monochromator. The reaction curve was fitted by a non-linear least square method to determine the first-order reaction rate constant. Next, the oxygen binding rate constant k ^O2 _on was calculated by dividing the obtained rate constant by the concentration of O ₂ .

[Measurement of oxygen dissociation rate constant k ^O2 _off ]
The oxygen dissociation rate was determined by the potassium ferricyanide method. A stopped flow apparatus was used for the measurement, and the probe light was passed through a monochromator. The absorbance change at 580 nm was fitted by a first-order rate equation under the condition of [K ₃ [Fe (CN) ₆ ]] >> [Mb]]. According to the following formula (1), k ^O2 _off was determined from the rate constant observed when K ₃ [Fe (CN) ₆ ] was in a large excess.

Here, k _ox is an oxidation rate constant from the deoxy body to the met body. When a steady-state approximation holds, the reaction follows a first-order rate constant, and the apparent rate constant k _obs can be expressed as in equation (2).

When the condition of k _ox [K ₃ [Fe (CN) ₆ ]] >> k ^O2 _on [O ₂ ] is satisfied, the oxygen dissociation rate constant k ^O2 _off can be calculated as follows.

[Measurement of _auto- oxidation rate constant k _auto ]
At 37 ° C., a UV-vis spectrum was measured every 30 minutes in the range of 500 to 650 nm using a natural myoglobin oxygen complex or a protein polymer (I-13) oxygen complex as a sample. The change in absorbance at 580 nm was plotted against time, and the autooxidation rate constant k _auto was calculated by fitting with the first-order rate equation. The obtained results are shown in Table 1.

  From the results shown in Table 1, the protein polymer (I-13) having the monomer unit of the protein monomer represented by the formula (V-13) or its positional isomer or a salt thereof has an affinity for oxygen superior to that of the natural type. It was confirmed that it had.

(Iii) Measurement of size exclusion chromatogram With respect to the protein polymer (I-13) obtained in Example 1, size exclusion chromatography was measured. A Superdex200 10 / 300GL column was used as the column. A 100 mM phosphate buffer (pH 7.0) was used as an elution solvent. The measurement was performed at a temperature of 4 ° C. and a flow rate of 0.5 mL / min. The result is shown in FIG. In FIG. 2a, the faster the eluted component, the larger the molecular weight. The elution amount of the myoglobin variant A125C monomer is 17.6 mL, and the elution amount of the myoglobin variant A125C dimer is 16.2 mL (not shown). The elution amount of protein polymer (I-13) was 9.3 mL. Therefore, it was confirmed that the protein polymer had a smaller elution amount than these myoglobin variant A125C monomers and dimers, and the molecular weight of these polymers was large. Furthermore, the protein polymer (I-13) was greatly chromatographed from the detection limit of 8 mL, and it was confirmed that the protein polymer (I-13) contained many large molecular weight components. In addition, since the common logarithm of molecular weight and the elution amount are in a proportional relationship, the molecular weight of the protein polymer (I-13) can be calculated roughly. From the molecular weight and monomer molecular weight, the degree of polymerization of the protein polymer (I-13) was calculated to be 33.

The crosslinked structure obtained in Example 1 (4) was measured for size exclusion chromatography using a Superdex200 10 / 300GL column. The result is shown in FIG. In FIG. 2 b, the solid line is a chart of the crosslinked structure of Example 1, and the broken line is a chart of the protein polymer (I-13) obtained in Example 1. From FIG. 2b, it can be confirmed that the structure of the present invention has a very large molecular weight mainly composed of components reaching the exclusion limit (elution amount: 7.5 mL, molecular weight: 1.3 × 10 ⁶ Da). It was.

  Moreover, about the crosslinked structure obtained in Example 1, size exclusion chromatography was measured using a KW405-4F column manufactured by Shodex instead of the Superdex200 10 / 300GL column. The result is shown in FIG. In FIG. 2c, a solid line is a chart of the crosslinked structure of Example 1, and a broken line is a chart of the protein polymer (I-13) obtained in Example 1. From FIG. 2c, it was confirmed that the structure of the present invention had a very large molecular weight.

(Iv) Measurement of Size Exclusion Chromatogram under Conditions with Changed Concentration For the protein polymer (I-13) obtained in Example 1, the polymer concentration was 10 μM, 100 μM, 500 μM and 1000 μM, respectively. The size exclusion chromatogram was measured in the same manner as (ii) above. The result is shown in FIG. From FIG. 3, it was confirmed that as the concentration of the polymer (I-13) was increased, the length of the protein polymer (I-13) was increased and the amount of the low molecular weight component of the protein polymer was decreased. . This indicates that the protein polymer (I-13) changes the molecular weight distribution depending on the concentration, and is understood to be governed by thermodynamic equilibrium.

(V) Atomic force microscope (AFM) measurement The sample used for AFM measurement was prepared as follows. The surface of the highly oriented sintered graphite (HOPG) substrate was wall-opened, and the wall-opening surface was an aqueous solution of the protein polymer obtained in Example 1 (3) (about 10 ⁻⁸ M, 0.05 M Tris buffer, The sample was immersed in pH 7.3 or 0.1 M phosphate buffer (pH 7.0) for several seconds. After the substrate was lifted from the solution, it was thoroughly washed with water, dried well in a desiccator containing calcium chloride at room temperature, and then set in an AFM measuring apparatus. The measurement was performed in a tapping mode, and a silicon single crystal having a scanning speed of 2 Hz and a radius of curvature of about 10 nm was used for the probe. An AFM image of the protein polymer (I-13) obtained in Example 1 (3) is shown in FIGS. 4 (a) and (b).

  4A shows an overall view of the protein polymer (I-13), and FIG. 4B shows a profile along the white line in (a). For this protein polymer, an image showing a linear polymer structure having a uniform height could be confirmed. In addition, a linear polymer structure having a length corresponding to several tens of monomers was also confirmed.

(Vi) Measurement of mass spectrum Moreover, the ESI-TOF-MS spectrum of the protein monomer (V-13) or its positional isomer obtained in Example 1 (3) is shown in FIG. A deconvoluted ESI-TOF-MS spectrum of V-13) is shown in FIG. The calculated molecular weight of the protein monomer (V-13) is 18260.9. From FIG. 5B, it was confirmed that the molecular weight of the protein monomer (V-13) or its positional isomer was consistent with the calculated value.

(Vii) Polyacrylamide gel electrophoresis (PAGE)
Cross-linked structure obtained in Example 1 (4) (7.2 × 10 ⁻⁷ g, 4.0 × 10 ⁻¹¹ mol in terms of heme) to sodium dodecyl sulfate (SDS) (8.0 × 10 ^After adding ⁻⁵ g, 2.8 × 10 ⁻⁷ mol) to denature the protein, electrophoresis was performed at 15 ° C. using a gel (manufactured by GE Healthcare, Phasgel Homogenius 12.5). The gel after electrophoresis was subjected to protein staining using Coomassie Brilliant Blue dye. The obtained electrophoresis results are shown in FIG.

  In FIG. 6, lane 1 is the protein polymer (I-13) obtained in Example 1 (3), and lanes 2 to 5 are from the left, 0.5 equivalent, 1 equivalent to the protein polymer (I-13), A crosslinked structure obtained by reacting with 0.0, 1.5 and 2.0 equivalents of hydrogen peroxide, Lane 6, reacts 1.5 equivalents of hydrogen peroxide against natural myoglobin The cross-linked natural myoglobin structure obtained in the above, Lane 7 is a marker using a standard protein. From FIG. 6, the crosslinked structure obtained by reacting 0.5 equivalent, 1.0 equivalent, 1.5 equivalent and 2.0 equivalent of hydrogen peroxide with respect to the protein polymer (I-13) is It was confirmed that a covalently crosslinked dimer was formed. The cross-linked myoglobin structure was produced according to O. M. Lardinois et al. (O. M. Lardinois, P. R. Ortiz de Montellano, J. Biol. Chem. 278, 36214 (2003)).

(Viii) Scanning electron microscope (SEM) measurement The surface of the highly oriented sintered graphite (HOPG) substrate was wall-opened, and the solution of the crosslinked structure obtained in Example 1 (4) was formed on the wall open surface. (10 nM, 5 μL, ultrapure aqueous solution) was added dropwise. The substrate was immersed in liquid nitrogen, the droplets were frozen, and the water was lyophilized under vacuum. After sufficiently drying, the substrate was set in an SEM measuring apparatus. The measurement was performed using a scanning electron microscope (S-5500, manufactured by Hitachi High-Tech) in a secondary electron detection mode and an acceleration voltage of 1.5 kV. The obtained SEM image is shown in FIG. 7 (a), 7 (b) and 7 (c) are images with different scales. As shown in FIG. 7A, it was confirmed that the crosslinked structure of the present invention was a sponge-like structure having a size of about several tens to several hundreds of nanometers. Moreover, as shown in FIG.7 (c), it has confirmed that the width | variety of the fiber which comprises the sponge-like structure of Example 1 was 4-9 nm. Considering the size of natural myoglobin, it was confirmed that the one-dimensional polymer having the protein monomer (V-13) as a monomer was produced by crosslinking.

(Ix) Oxygen complex of the crosslinked structure of Example 1 The UV-visible absorption spectrum of the crosslinked structure obtained in Example 1 (4) is shown in FIG. 8 (a) and in Example 1 (5). The ultraviolet-visible absorption spectrum of the obtained oxygen complex of the structure is shown in FIG. As shown in FIG. 8 (a), the UV-visible absorption spectrum of the crosslinked structure is in good agreement with the UV-visible absorption spectrum of natural myoglobin, and iron porphyrin is correctly incorporated into the protein in the structure. It was confirmed that Further, as shown in FIG. 8B, the UV-visible absorption spectrum of the oxygen complex of the structure is in good agreement with the UV-visible absorption spectrum of the natural myoglobin oxygen complex, and the structure has an oxygen storage capacity. It was confirmed that it had.

  The structure of the present invention can also be used as an assembly of enzymes.

SEQ ID NO: 1 cytochrome b ₅₆₂
SEQ ID NO: 2 Sperm whale wild-type myoglobin
SEQ ID NO: 3 Horseradish peroxidase SEQ ID NO: 4 hemoglobin α subunit (human)
SEQ ID NO: 5 hemoglobin β subunit (human)

Claims (6)

A protein polymer or a salt thereof is a structure crosslinked by peracid treatment,
A structure in which the protein polymer or a salt thereof is a protein polymer represented by the general formula (IA) or the general formula (IB) or a salt thereof.
In the formula (IA) or (IB),
R ¹ , R ² , R ³ and R ⁴ each independently represents a hydrogen atom, a lower alkyl group, a lower alkyl group or a lower alkenyl group substituted with a halogen atom,
Y is a group represented by -Z1-Y1-Z2-Y2- or -Z1-Y1-, wherein Z1 is a group bonded to -CO- in formula (IA) and formula (IB). Means
Z1 is selected from the group consisting of O, NH and S;
Y1 and Y2 are each independently formula _{- (CH 2) n1 -,} - (C 6 H 4) n5 -, - (CH 2) n1 - (C 6 H 4) n5 -, - (C 6 H _{4) n5 - (CH 2)} n1 -, - (CH 2) n1 - (C 6 H 4) n5 - (CH 2) n2 -, - (CH 2) n1 -O- (CH 2) n2 -, - _{(CH 2) n1 -O- (CH} 2) n2 -O- (CH 2) n3 - or _{- (CH 2) n1 -O- (} CH 2) n2 -O- (CH 2) n3 -O- (CH ₂ ) a group represented by _n4 —, wherein n1, n2, n3, n4 and n5 each independently represents an integer of 1 to 20, and the (C ₆ H ₄ ) represents one or more substituents May have a group,
Z2 is selected from the group consisting of NHCO, CONH, NH, O, N = N, N = CH, CH = N, C = C;
M is selected from the group consisting of Fe ²⁺ , Fe ³⁺ , Zn ²⁺ , Co ²⁺ , Co ³⁺ , Mn ²⁺ and Mn ³⁺ ;
m and n are each an integer, 1 ≦ m + n ≦ 1000,
X means X in the hemoprotein mutant, and the hemoprotein mutant is represented by the formula (II);
The mutant is obtained by replacing one amino acid residue with a cysteine residue in the amino acid sequence of a natural heme protein. In the mutant, -SH means a side chain thiol group of the cysteine residue, Heme means natural heme, and formula HS-X means an apo form of the mutant.   The structure according to claim 1, wherein the natural hemoprotein is cytochrome, hemoglobin, myoglobin, or peroxidase. In the formula (IA) or (IB), R ¹ , R ² , R ³ and R ⁴ each independently represents a lower alkyl group or a lower alkenyl group,
Y is a group represented by -Z1-Y1-Z2-Y2- or -Z1-Y1-, wherein Z1 is a group bonded to -CO- in formula (IA) and formula (IB). Means
Z1 is selected from the group consisting of O, NH and S;
Y1 and Y2 are each independently formula _{- (CH 2) n1 -,} - (C 6 H 4) n5 -, - (CH 2) n1 - (C 6 H 4) n5 -, - (CH 2) _{n1 -O- (CH 2) n2 -} , - (CH 2) n1 -O- (CH 2) n2 -O- (CH 2) n3 - or _{- (CH 2) n1 -O- (} CH 2) n2 - A group represented by O— (CH ₂ ) _n3 —O— (CH ₂ ) _n4 —, wherein n1, n2, n3, n4 and n5 each independently represents an integer of 1 to 20,
The structure according to claim 1 or 2, wherein Z2 is selected from the group consisting of NHCO, CONH, NH, O, N = N, N = CH, CH = N, and C = C. A method for producing a structure in which a protein polymer or a salt thereof is crosslinked,
The protein polymer represented by the general formula (IA) or the general formula (IB) is subjected to a condition for forming a covalent bond between molecules, and the protein polymer represented by the general formula (IA) or the general formula (IB). Or the salt is crosslinked between molecules to obtain the structure,
The condition for forming the covalent bond is a condition for generating a radical,
A production method wherein the condition for generating the radical is contact with a peracid.
In the formula (IA) or (IB),
R ¹ , R ² , R ³ and R ⁴ each independently represents a hydrogen atom, a lower alkyl group, a lower alkyl group or a lower alkenyl group substituted with a halogen atom,
Y is a group represented by -Z1-Y1-Z2-Y2- or -Z1-Y1-, wherein Z1 is a group bonded to -CO- in formula (IA) and formula (IB). Means
Z1 is selected from the group consisting of O, NH and S;
Y1 and Y2 are each independently formula _{- (CH 2) n1 -,} - (C 6 H 4) n5 -, - (CH 2) n1 - (C 6 H 4) n5 -, - (C 6 H _{4) n5 - (CH 2)} n1 -, - (CH 2) n1 - (C 6 H 4) n5 - (CH 2) n2 -, - (CH 2) n1 -O- (CH 2) n2 -, - _{(CH 2) n1 -O- (CH} 2) n2 -O- (CH 2) n3 - or _{- (CH 2) n1 -O- (} CH 2) n2 -O- (CH 2) n3 -O- (CH ₂ ) a group represented by _n4 —, wherein n1, n2, n3, n4 and n5 each independently represents an integer of 1 to 20, and the (C ₆ H ₄ ) represents one or more substituents May have a group,
Z2 is selected from the group consisting of NHCO, CONH, NH, O, N = N, N = CH, CH = N, C = C;
M is selected from the group consisting of Fe ²⁺ , Fe ³⁺ , Zn ²⁺ , Co ²⁺ , Co ³⁺ , Mn ²⁺ and Mn ³⁺ ;
m and n are each an integer, 1 ≦ m + n ≦ 1000,
X means X in the hemoprotein mutant, and the hemoprotein mutant is represented by the formula (II);
The mutant is obtained by replacing one amino acid residue with a cysteine residue in the amino acid sequence of a natural heme protein. In the mutant, -SH means a side chain thiol group of the cysteine residue, Heme means natural heme, and formula HS-X means an apo form of the mutant. The method according to claim 4 , wherein the peracid is selected from the group consisting of hydrogen peroxide, peracetic acid, metachloroperbenzoic acid and cumene hydroperoxide. The biological device containing the structure in any one of Claims 1-3.