Method for stabilizing heme protein

Abstract

The purpose of the present invention is to provide a method and a reagent for stabilizing a heme protein suitable for use in the fields of biochemistry and analytical chemistry for applications including clinical diagnostic agents.　Disclosed is a method for stabilizing a heme protein, the method being characterized by allowing a hydantoin derivative to coexist with the heme protein.

Description

Method for stabilizing heme proteins

[0001] The present invention relates to a method for stabilizing a hemoprotein, and more particularly to a method for stabilizing a hemoprotein, which comprises causing the hemoprotein to coexist with a hydantoin derivative.

[0002] Catalase (EC 1.11.1.6) is a hemoprotein with protoheme as its functional group, and is an enzyme that catalyzes the decomposition of hydrogen peroxide. Catalase is used in reagents for measuring triglycerides and creatinine to eliminate hydrogen peroxide derived from substances not being measured.

[0003] Patent Document 1 describes that a glycated protein measurement reagent containing catalase and 4-aminoantipyrine is stabilized while maintaining the activity of catalase by adding a specific chelating agent to the measurement reagent.

[0004] Patent Publication No. 2021-7341

[0005] Although Patent Document 1 describes the discovery of a specific chelating agent that can stabilize a measurement reagent while maintaining the activity of catalase, which is normally inactivated by chelating action, it does not mention preventing the decomposition or denaturation of catalase and thereby increasing the stability of catalase. On the other hand, the present inventors have discovered a problem in that catalase is particularly susceptible to decomposition and denaturation in water, and that long-term storage of a reagent containing catalase results in the inactivation of catalase.

[0006] An object of the present invention is to provide a method for stabilizing a hemoprotein such as catalase.

[0007] The present inventors have conducted extensive research to solve the above-mentioned problems and have found that hydantoin derivatives stabilize hemoproteins. Based on this finding, the present inventors have conducted further extensive research and have completed the present invention.

[0008] The present invention encompasses the following aspects: (Item 1) A method for stabilizing a hemoprotein, characterized by allowing a hydantoin derivative to coexist. (Item 2) The method according to Item 1, wherein the hydantoin derivative is a compound represented by the following formula (1): (In the formula, R1 to R3 are independently hydrogen or a substituent selected from the group consisting of an alkyl group, a hydroxyalkyl group, an amino group, a hydroxycarbonylamino group, a formylamino group, an acylamino group, a carbamoyl group, a carboxyl group, an aldehyde group, an acyl group, a hydroxyl group, a sulfone group, and a phenyl group, or a residue of a compound having at least one of the above substituents; R2 and R3 may together form an oxo group or a ring; and R1, R2, and R3 are each a group represented by formula (2): (Item 3) The hydantoin derivative may be N,N''-methylenebis[N'-[3-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]urea] (imidazolidinyl urea), N,N'-bis(hydroxymethyl)-N-[1,3-bis(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]urea (diazolidinyl urea), 1,3-bis(hydroxymethyl)-5,5-dimethylhydantoin (DMD Item 4: The method according to Item 2, wherein in formula (1), R1 is hydrogen or a hydroxyalkyl group. (Item 5): The method according to Item 2, wherein in formula (1), R2 is an alkyl group or an amino group, or a residue of a compound having an alkyl group and an amino group or an amide group (including a peptide bond and a urea bond). (Item 6): The method according to any one of Items 2, 4, and 5, wherein in formula (1), R3 is hydrogen or an alkyl group. (Item 7): The method according to any one of Items 2, 4, and 5, wherein in formula (1), R3 is hydrogen or an alkyl group. (Item 8): The method according to Item 3, wherein in formula (1), R4 is hydrogen or an alkyl group. (Item 8) The method according to any one of Items 1 to 6, wherein the hydantoin derivative is an imidazolidinyl urea represented by formula (1-2): (Item 9) The method according to any one of Items 1 to 6, wherein the hydantoin derivative is a diazolidinyl urea represented by formula (1-3): The method of any one of Items 1 to 6, wherein the hydantoin derivative is DMDM ​​hydantoin represented by the formula: (Item 10) The method of any one of Items 1 to 9, wherein the hydantoin derivative is contained at 0.16 mM or more in a reagent containing a hemoprotein. (Item 11) The method of any one of Items 1 to 10, wherein the hemoprotein is catalase. (Item 12) The method of Items 10 or 11, wherein the concentration of catalase in the reagent is 10 to 500 KU / L. (Item 13) The method of any one of Items 10 to 12, wherein the reagent is used for measuring a biological component. (Item 14) The method of Item 13, wherein the biological component is at least one selected from the group consisting of creatinine, triglycerides, inorganic phosphorus, creatine, cholesterol esters, sialic acid, α-amylase, GOT, GPT, guarase, and phospholipids. (Item 15) The method of Items 13 or 14, wherein the biological component is creatinine.

[0009] (Item 16) A method for colorimetrically measuring a biological component by reacting a sample containing or suspected to contain a biological component to be measured with at least one enzyme and then carrying out a color development reaction, characterized in that a hemoprotein and a hydantoin derivative are allowed to coexist in a measurement reagent. (Item 17) The method according to Item 16, wherein the hydantoin derivative is a compound represented by the following formula (1): (In the formula, R1 to R3 are independently hydrogen or a substituent selected from the group consisting of an alkyl group, a hydroxyalkyl group, an amino group, a hydroxycarbonylamino group, a formylamino group, an acylamino group, a carbamoyl group, a carboxyl group, an aldehyde group, an acyl group, a hydroxyl group, a sulfone group, and a phenyl group, or a residue of a compound having at least one of the above substituents; R2 and R3 may together form an oxo group or a ring; and R1, R2, and R3 are each a group represented by formula (2): may be linked to R1, R2, and R3 of a compound represented by the formula:

[0010] (Item 18) A reagent or kit for measuring a biological component, comprising at least the following (a) to (f) as constituent elements: (a) a hydantoin derivative, (b) a catalase, (c) a peroxidase, (d) one or more enzymes other than catalase and peroxidase, (e) a buffer, and (f) a color developer. (Item 19) The reagent or kit for measuring a biological component according to Item 18, wherein the hydantoin derivative is a compound represented by the following formula (1): (In the formula, R1 to R3 are independently hydrogen or a substituent selected from the group consisting of an alkyl group, a hydroxyalkyl group, an amino group, a hydroxycarbonylamino group, a formylamino group, an acylamino group, a carbamoyl group, a carboxyl group, an aldehyde group, an acyl group, a hydroxyl group, a sulfone group, and a phenyl group, or a residue of a compound having at least one of the above substituents; R2 and R3 may together form an oxo group or a ring; and R1, R2, and R3 are each a group represented by formula (2): (Item 20) The reagent or kit for measuring a biological component according to Item 18 or 19, wherein the (d) one or more enzymes other than catalase and peroxidase are at least one selected from the group consisting of ascorbic acid oxidase, sarcosine oxidase, creatine amidinohydrolase, and creatinine amidohydrolase. (Item 21) The reagent or kit for measuring a biological component according to any of Item 18 to 20, wherein the (e) buffer is at least one selected from the group consisting of Tris buffer, citrate buffer, borate buffer, phosphate buffer, MES, Bis-Tris, ADA, ACES, BES, PIPES, MOPS, TES, HEPES, Tricine, Bicine, POPSO, TAPS, CHES, and CAPS. (Item 22) The color former (f) is N-ethyl-N-(3-sulfopropyl)-m-anisidine, aniline, N,N-dimethylaniline, N,N-diethylaniline, N,N-diethyl-m-toludine, N,N-dimethyl-m-anisidine, N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine, N-ethyl-N-(β-hydroxyethyl)-m-toluidine, N-ethyl-N-(2-hydroxy-3-sulfoethyl)-m-toluidine, N-ethyl-N-sulfopropyl- 22. The reagent or kit for measuring a biological component according to any one of Items 18 to 21, wherein the phenol is any one of m-toluidine, N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine, phenol, p-chlorophenol, 2,4-dichlorophenol, 2,4-dibromophenol, and 2,3,4-trichlorophenol.

[0011] The present invention provides a method for stabilizing a hemoprotein, for example, a method for stabilizing catalase contained in a reagent for measuring a biological component. Currently, most clinical diagnostic reagents are liquid reagents, and a liquid clinical diagnostic reagent that is stable for a long period of time is extremely useful.

[0012] The present invention will be described in further detail below while illustrating embodiments of the present invention, but the present invention is not limited thereto. All non-patent documents and patent documents described in this specification are incorporated herein by reference. In addition, the term "to" in this specification means "at least or equal to, at most or equal to," and for example, when the specification states "X to Y," it means "at least X and at most Y." In addition, the term "and / or" in this specification means any one or any possible combination of two or more of the listed elements. In addition, in this specification, the singular form should be understood to include the plural form, unless otherwise specified.

[0013] (Method for stabilizing hemoproteins) One embodiment of the present invention provides a method for stabilizing hemoproteins by allowing a hydantoin derivative to coexist with the hemoproteins. Reagents (especially reagents for measuring biological components) to which this stabilization method is applied are extremely useful.

[0014] The method for stabilizing a hemoprotein includes adding a hydantoin derivative to a reagent containing the hemoprotein. The hydantoin derivative is preferably a compound represented by the following formula (1): (wherein R1 to R3 are independently hydrogen, an alkyl group, a hydroxyalkyl group, an amino group, a hydroxycarbonylamino group (-NH-COOH), a formylamino group (-NH-CHO), an acylamino group, a carbamoyl group (-CO-NH ２ ), carboxyl group, aldehyde group (-CHO), acyl group, hydroxyl group, sulfonic acid group (-SO ３ R1, R2, and R3 are each a substituent selected from the group consisting of a phenyl group, a phenyl group, or a residue of a compound having at least one of the above substituents (for example, a group formed by combining two or more of the above substituents), and R2 and R3 may together form an oxo group (=O) or a ring, and R1, R2, and R3 are each a group represented by formula (2): may be linked to R1, R2, and R3 of a compound represented by the formula:

[0015] In this specification, an alkyl group refers to a monovalent group obtained by removing one hydrogen atom from a linear or branched saturated hydrocarbon. The number of carbon atoms in the alkyl group is not particularly limited, but is, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 6, and particularly preferably 1 to 4. Specific examples of alkyl include methyl, ethyl, propyl (n-propyl, i-propyl), butyl (e.g., n-butyl, i-butyl, s-butyl, t-butyl), pentyl (e.g., n-pentyl, i-pentyl, neopentyl), hexyl, heptyl, octyl (e.g., n-octyl, 2-ethylhexyl), nonyl, and decyl.

[0016] As used herein, the term "hydroxyalkyl group" refers to an alkyl group substituted with one or more hydroxyl groups. Specific examples of hydroxyalkyl groups include hydroxymethyl, hydroxyethyl (1-hydroxyethyl, 2-hydroxyethyl), hydroxypropyl (e.g., 3-hydroxypropyl), hydroxybutyl (e.g., 4-hydroxybutyl), dihydroxymethyl, dihydroxyethyl (1,1-dihydroxyethyl, 1,2-dihydroxyethyl, 2,2-dihydroxyethyl), dihydroxypropyl (e.g., 2,3-dihydroxypropyl), dihydroxybutyl (e.g., 3,4-dihydroxybutyl), and the like.

[0017] In this specification, the term "acyl group" refers to a group represented by -CO-R. R is usually a hydrocarbon group, and specific examples thereof include an alkyl group, a cycloalkyl group, an aryl group, and an aralkyl group. Specific examples of acyl include acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, and decanoyl.

[0018] In this specification, the ring may be a hydrocarbon ring or a heterocyclic ring (for example, a ring containing one or more heteroatoms selected from the group consisting of N, O, and S as ring-constituting atoms), may be a saturated ring or an unsaturated ring, and may be a monocyclic ring or a fused ring. Specific examples of the ring include cycloalkane rings such as a cyclopentane ring, a cyclohexane ring, a cycloheptane ring, and a cyclooctane ring; arene rings such as a benzene ring and a naphthalene ring; non-aromatic heterocyclic rings such as a piperidine ring, a piperazine ring, a morpholine ring, and a tetrahydropyran ring; and heteroarene rings such as a furan ring, a thiophene ring, a pyrrole ring, and a pyridine ring. The ring may be substituted with one or more substituents such as those exemplified for R1 to R3.

[0019] (Hemoprotein) A hemoprotein is a protein containing heme. Heme refers to a complex consisting of a divalent iron atom and porphyrin. Porphyrin is a cyclic planar structure composed of four pyrrole molecules, and heme has a divalent iron atom at the center of this cyclic planar structure. Examples of hemoproteins include hemoglobin, which is an oxygen carrier, cytochromes involved in the electron transport system, catalase and peroxidase, which have enzymatic activity. Hemoproteins may or may not have enzymatic activity, but those with enzymatic activity are preferred. Examples of hemoproteins with enzymatic activity include catalase and peroxidase.

[0020] (Catalase) Catalase is particularly preferred as the hemoprotein. Catalase (EC 1.11.1.6) is a hemoprotein having protoheme and is an enzyme that catalyzes the reaction of decomposing hydrogen peroxide. There are no particular limitations on the catalase as long as it can achieve the effects of the present invention, and catalase derived from any microorganism (fungi, bacteria, archaea, etc.) can be used. For example, the catalase used in the present invention may be selected from the group consisting of Podospora, Neurospora, Cladosporium, Emericella, Pleurotus, Deinococcus, Escherichia, Salmonella, Pseudomonas, Bacillus, Mycobacterium, Botryothionia, and the like. The catalase may be derived from a microorganism of the genus Arthrobacterium, ... From the viewpoint of making it easier to obtain the effects of the present invention, it is preferable to use a catalase derived from a microorganism of the genus Corynebacterium or Arthrobacter.

[0021] The catalase used in the present invention may be a catalase of any size, for example, a catalase having a molecular weight (Mw) of about 50,000 or more (e.g., 50,000 to 90,000). In one embodiment, the catalase used in the present invention may be a catalase having a molecular weight of 75,000 or less (or less), a catalase having a molecular weight of 65,000 or less, or even a catalase having a molecular weight of 60,000 or less. By using catalases with such relatively small molecular weights, even greater effects can be expected. The molecular weight of catalase can be measured by mass spectrometry, specifically MALDI-TOF MS analysis.

[0022] The catalase used in the present invention has an optimum pH of preferably 6.5, more preferably 7, and even more preferably 7.2, and an optimum pH of preferably 9, more preferably 8.8, and even more preferably 8.5. In one embodiment, the optimum pH is preferably 6.5 to 9, and more preferably 7.2 to 8.5.

[0023] The catalases used in the method of the present invention may be a single catalase or a mixture of two or more catalases. When two or more catalases are used, they may be derived from the same or different microorganisms.

[0024] Various catalases derived from these microorganisms are commercially available, and commercially available products can be preferably used. Alternatively, catalases can be obtained by culturing microorganisms and purifying them from the culture by conventional methods well known to those skilled in the art.

[0025] (Hydantoin Derivative) The hydantoin derivative is not particularly limited as long as it has a hydantoin skeleton (2,4-imidazolidinedione skeleton). In one embodiment, the hydantoin derivative preferably has a structure represented by the following formula (1): (wherein R1 to R3 have the same meanings as defined above.)

[0026] Without being bound by any particular theory, it is believed that the hydantoin derivative acts as a preservative and prevents the enzyme from being decomposed by microorganisms. Also, without being bound by any particular theory, it is believed that the hydrogen atoms contained in the hydantoin derivative form hydrogen bonds with the unshared electron pairs of the hemoprotein, thereby preventing the denaturation of the hemoprotein.

[0027] In formula (1), R1 is preferably hydrogen or a substituent selected from the group consisting of an alkyl group, a hydroxyalkyl group, an amino group, a hydroxycarbonylamino group, a formylamino group, an acylamino group, a carbamoyl group, a carboxyl group, an aldehyde group, an acyl group, a hydroxyl group, a sulfone group, and a phenyl group, more preferably hydrogen, an alkyl group, a hydroxyalkyl group, an acyl group, or a phenyl group, and even more preferably hydrogen or a hydroxyalkyl group (e.g., a hydroxy C group such as a hydroxymethyl group). １－４ alkyl group).

[0028] In formula (1), R2 is preferably hydrogen or a substituent selected from the group consisting of an alkyl group, a hydroxyalkyl group, an amino group, a hydroxycarbonylamino group, a formylamino group, an acylamino group, a carbamoyl group, a carboxyl group, an aldehyde group, an acyl group, a hydroxyl group, a sulfone group, a phenyl group, and a group represented by formula (2), or a compound having at least one of the above substituents, more preferably an alkyl group, a phenyl group, or an amino group, or a residue of a compound having an alkyl group and an amino group or an amide group (including a peptide bond and a urea bond), even more preferably an alkyl group or an amino group, or a residue of a compound having an alkyl group and an amino group or an amide group (including a peptide bond and a urea bond). In one embodiment, R2 is an alkyl group (e.g., a C １－４ Preferably, R4 and R5 are independently selected from those listed for R1 to R3. More preferably, R4 is -CO-NH-R6. Preferably, R6 is selected from those listed for R1 to R3. R6 is a hydroxyalkyl group (e.g., a hydroxy C group such as a hydroxymethyl group).１－４ alkyl group) or R5 is preferably hydrogen or hydroxyalkyl (e.g., hydroxy C such as hydroxymethyl group). １－４ It is more preferable that the aryl group is an alkyl group.

[0029] In formula (1), R3 is preferably a substituent selected from the group consisting of hydrogen, an alkyl group, a hydroxyalkyl group, an amino group, a hydroxycarbonylamino group, a formylamino group, an acylamino group, a carbamoyl group, a carboxyl group, an aldehyde group, an acyl group, a hydroxyl group, a sulfone group, and a phenyl group, and more preferably hydrogen or an alkyl group (e.g., a C １－４ alkyl group).

[0030] Hydantoin is a heterocyclic compound, structurally a cyclic condensation product of glycolic acid and urea. Hydantoin derivatives are not particularly limited as long as they do not inhibit the effects of the present invention, and examples include N-halogenated hydantoin derivatives such as phenytoin and dantrolene, which are used as pharmaceuticals, 1-bromo-3-chloro-5,5-dimethylhydantoin, which is used as a halogenating agent (reaction reagent), and 1,3-dibromo-5,5-dimethylhydantoin, which is used as a disinfectant. Further examples of hydantoin derivatives include the compounds shown in the table below.

[0031] In one preferred embodiment of the present invention, the compound of formula (1) is imidazolidinyl urea.

[0032] In one preferred embodiment of the present invention, the compound of formula (1) is diazolidinyl urea.

[0033] In one preferred embodiment of the present invention, the compound of formula (1) is DMDM ​​hydantoin.

[0034] (Method for Measuring a Biological Component) In one embodiment of the present invention, the method for measuring a biological component comprises reacting a sample containing or thought to contain the biological component to be measured with at least one enzyme, followed by a colorimetric measurement of the biological component by carrying out a color reaction, characterized in that a hemoprotein and a hydantoin derivative are allowed to coexist in the reagent.

[0035] The biological component is not particularly limited, but examples thereof include creatinine, triglycerides, inorganic phosphorus, creatine, cholesterol esters, sialic acid, α-amylase, GOT, GPT, guaiase, phospholipids, etc. The biological component may be one type alone or a combination of two or more types. The biological component is preferably creatinine.

[0036] (Reagent or Kit for Measuring Biological Component) The reagent or kit for measuring a biological component of the present invention (hereinafter also simply referred to as "reagent" or "kit") contains at least the following (1) to (6), and preferably contains (1) a hydantoin derivative and (2) a hemoprotein in the same composition: (1) a hydantoin derivative, preferably a compound represented by formula (1); (2) a hemoprotein, preferably catalase; (3) a peroxidase; (4) one or more enzymes other than catalase and peroxidase; (5) a buffer; and (6) a color developer.

[0037] The reagent is preferably a reagent (or kit) configured to be applicable to a general-purpose automated analyzer (e.g., Hitachi 7170 automated analyzer). Examples of such a form include a reagent (or kit) configured by combining a dry preparation produced by means such as lyophilization with a dissolving solution. A liquid reagent is preferred, such as a liquid reagent divided into two separate packages (hereinafter also referred to as a two-reagent liquid reagent). In this method, a first type of reagent (hereinafter also referred to as the first reagent) is first added to a sample and allowed to react for a certain period of time, and then a second type of reagent (hereinafter also referred to as the second reagent) is further added and allowed to react. The target component can be quantified by measuring the change in absorbance during this period.

[0038] The reagent (or kit) can take various forms, including the above-mentioned forms, a kit called a dry system in which an enzyme or the like is supported on a suitable carrier, and a method using a sensor.

[0039] According to a preferred embodiment of the present invention, the reagent is a reagent for measuring creatinine. The composition of the reagent for measuring creatinine is not particularly limited as long as it does not inhibit the effects of the present invention. For example, it is preferable that the reagent contains at least the following (1) to (6), and that (1) a hydantoin derivative and (2) catalase are contained in the same composition: (1) a hydantoin derivative, preferably a compound represented by formula (1), (2) catalase, (3) peroxidase, (4) one or more enzymes other than catalase and peroxidase, (5) a buffer, and (6) a color developer.

[0040] In the creatinine measurement reagent, (5) the buffer is preferably in the form of a buffer dissolved in a solvent such as water, and such a buffer preferably contains (1) a hydantoin derivative and (2) catalase. Examples of the buffer (or buffer solution) include Tris buffer (or Tris buffer solution), citrate buffer (or citrate buffer solution), phosphate buffer (or phosphate buffer solution), borate buffer (or borate buffer solution), carbonate buffer (or carbonate buffer solution), and GOOD's buffer (or GOOD's buffer solution). There are no particular restrictions on the amount used, the set pH, the form of addition, etc. All of these can be obtained commercially.

[0041] Examples of GOOD's buffers (or GOOD's buffer solutions) include 3-morpholinopropanesulfonic acid (MOPS), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 2-morpholinoethanesulfonic acid (MES), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), N-tris(hydroxymethyl)methyl-2-aminomethanesulfonic acid (TES), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), Examples include 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (EPPS), 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPSO), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) (POPSO), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPSO), N-(2-acetamido)iminodiacetic acid (ADA), N,N-bis(2-hydroxyethyl)glycine (Bicine), and N-[tris(hydroxymethyl)methyl]glycine (Tricine).

[0042] The type and concentration of the buffer in the reagent are not particularly limited. Preferably, the buffer is contained in an amount adjusted so that the concentration in the reagent is 0 to 500 mM, preferably 5 to 100 mM, more preferably 10 to 75 mM, and even more preferably 20 to 50 mM.

[0043] The concentration of the hydantoin derivative in the reagent is not particularly limited. Preferably, the hydantoin derivative is contained in an amount adjusted so that the concentration in the reagent is 0 to 10.0 mM, preferably 0.05 to 8.0 mM, more preferably 0.10 to 5.0 mM, and even more preferably 0.16 to 4.0 mM. In one embodiment, the concentration of the hydantoin derivative in the reagent containing a hemoprotein is 0.16 mM or more.

[0044] When catalase is used as the hemoprotein, the concentration of catalase in the reagent is not particularly limited. Preferably, the catalase is contained in an amount adjusted so that the concentration of catalase in the reagent is 10 to 500 KU / L, preferably 15 to 370 KU / L, more preferably 50 to 350 KU / L, and even more preferably 100 to 300 KU / L.

[0045] (Color former) Any type of color former may be used in the present invention as long as it reacts with hydrogen peroxide to form a dye and develop a color. Examples include a combination of a hydrogen donor and a coupler, a leuco compound, a tetrazolium salt, etc. From the viewpoint of more effectively achieving the effects of the present invention, it is preferable to use a combination of a hydrogen donor and a coupler as the color former. There are no particular restrictions on the amount of the color former used or the form of addition. All of these can be obtained commercially.

[0046] A typical example of a combination of a hydrogen donor and a coupler involves the oxidative condensation of a Trinder reagent (hydrogen donor) and a coupler with hydrogen peroxide in the presence of peroxidase to form a dye. Examples of hydrogen donors used as Trinder reagents include phenol, phenol derivatives (p-chlorophenol, 2,4-dichlorophenol, 2,4-dibromophenol, 2,3,4-trichlorophenol, etc.), aniline, aniline derivatives, naphthol, naphthol derivatives, naphthylamine, naphthylamine derivatives, and ethylenediamine derivatives, and these can be suitably used in the present invention. Aniline, aniline derivatives, and ethylenediamine derivatives (particularly aniline derivatives) are preferred.

[0047] Examples of the hydrogen donor include N,N-dimethylaniline, N,N-diethylaniline, N,N-diethyl-m-toluidine, N,N-dimethyl-3-methoxyaniline (N,N-dimethyl-m-anisidine), N-ethyl-N-(β-hydroxyethyl)-m-toluidine, N-ethyl-N-(2-hydroxy-3-sulfoethyl)-m-toluidine, N-ethyl-N-sulfopropyl-m-toluidine, and N-ethyl-N-sulfopropyl-3- Methoxyaniline, N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline (N-ethyl-N-(3-sulfopropyl)-m-anisidine), N-ethyl-N-sulfopropylaniline, N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline, N-sulfopropyl-3,5-dimethoxyaniline, N-ethyl-N-sulfopropyl-3,5-dimethylaniline, N-ethyl-N-sulfopropyl-3-methylaniline, N-ethyl aniline derivatives such as N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine), N-sulfopropylaniline, and N-(2-hydroxy-3-sulfopropyl)-2,5-dimethylaniline; N-ethyl-N-(3-methylphenyl)-N'-succinylethylenediamine, and N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine. It is preferred to use these hydrogen donors in combination with a coupler.

[0048] Examples of couplers that can be suitably used in the present invention include 4-aminoantipyrine (4-AA), aminoantipyrine derivatives, vanillindiaminesulfonic acid, methylbenzthiazolinone hydrazone (MBTH), and sulfonated methylbenzthiazolinone hydrazone (SMBTH). Preferably, the coupler is 4-aminoantipyrine.

[0049] Examples of leuco compounds include triphenylmethane derivatives, phenothiazine derivatives, diphenylamine derivatives, etc. Specific examples include 4,4'-benzylidenebis(N,N-dimethylaniline), 4,4'-bis[N-ethyl-N-(3-sulfopropylamino)-2,6-dimethylphenyl]methane, 1-(ethylaminothiocarbonyl)-2-(3,5-dimethoxy-4-hydroxyphenyl)-4,5-bis(4-diethylaminophenyl)imidazole, 4,4'-bis(dimethylamino)diphenylamine, N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine salt (DA64), and 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine salt (DA67).

[0050] Examples of tetrazolium salts include 2,3,5-triphenyltetrazolium salt, 2,5-diphenyl-3-(1-naphthyl)-2H-tetrazolium salt, 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium] salt, and 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium] salt. bis(2,5-diphenyl-2H-tetrazolium) salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium salt, 3,3'-(1,1'-biphenyl-4,4'-diyl)-bis(2,5-diphenyl-2H-tetrazolium) salt, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium salt.

[0051] The creatinine measurement reagent can be used for measuring creatinine, but is not limited to the following. First, creatine produced by the reaction of creatinine amidinohydrolase using creatinine as a substrate is further reacted with creatine amidohydrolase to produce sarcosine. Next, hydrogen peroxide is produced from sarcosine using sarcosine oxidase. The produced hydrogen peroxide is then quantified using a peroxidase-coloring agent system to measure the creatinine concentration.

[0052] The creatinine measurement reagent may contain ascorbic acid oxidase and / or bilirubin oxidase, which can be used to eliminate bioreductive substances, such as ascorbic acid and / or bilirubin, that tend to affect creatinine measurement results.

[0053] In a preferred embodiment of the present invention, the creatinine measurement reagent or kit preferably comprises a first reagent, which is a buffer solution containing a hydantoin derivative, preferably a compound represented by formula (1), and a hemoprotein, preferably catalase, and a second reagent, which is a buffer solution containing creatinine amidohydrolase and a color developer, such as Trinder's reagent. The first reagent preferably further comprises ascorbic acid oxidase, sarcosine oxidase, and / or creatine amidinohydrolase. In one embodiment, the creatine amidinohydrolase contained in the first reagent acts on creatine in a sample to produce sarcosine, and the sarcosine oxidase contained in the first reagent acts on the produced sarcosine to produce hydrogen peroxide. The catalase contained in the first reagent acts on the produced hydrogen peroxide, thereby eliminating the hydrogen peroxide derived from creatine and suppressing its effect on creatinine measurement. In one embodiment, after the first reagent is added to eliminate the hydrogen peroxide derived from creatine, the second reagent is added to generate hydrogen peroxide derived from creatinine, and the generated hydrogen peroxide is reacted with a color developer, thereby enabling accurate quantification of the hydrogen peroxide derived from creatinine. The creatinine measurement reagent kit may further include a sample collection tool, an instruction manual, etc., in addition to the bottle containing the first reagent and the bottle containing the second reagent.

[0054] The present invention will be specifically described below with reference to examples, although the present invention is not particularly limited by these examples.

[0055] Example 1: Confirmation of catalase activity by addition of imidazolidinyl urea Imidazolidinyl urea was added to the first reagent of the creatinine measurement reagent described below at a concentration of 0.16 to 1.93 mM. As a comparative example, a first reagent without added imidazolidinyl urea was also prepared, and catalase activity was measured.

[0056] (Preparation of Reagents) Creatinine Measurement Reagents First Reagent MOPS 25.0 mmol / L (pH 7.7) Ascorbic acid oxidase (ASO-311, manufactured by Toyobo Co., Ltd.) 2.5 KU / L Sarcosine oxidase (SAO-351, manufactured by Toyobo Co., Ltd.) 8.0 KU / L Creatine amidinohydrolase (CRH-221, manufactured by Toyobo Co., Ltd.) 28.0 KU / L N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline 0.14 g / L Catalase (CAO-519, Mw 53,000, manufactured by Toyobo Co., Ltd.) 120 KU / L

[0057] For the measurement, two types of the first reagent were used: one that had been stored at 4°C and the other that had been stored at 35°C for one week.

[0058] (Measurement method) Catalase (CAO) (Principle) The amount of hydrogen peroxide reduced is measured by the titanium color reaction method. (Definition) One unit (U) is the amount of enzyme that decomposes 1 micromole of hydrogen peroxide per minute under the following conditions. (Reagents) (A) 10 mM phosphate buffer, pH 7.0 (B) H ２ O ２ Solution: 16 mM [0.182 mL of 30% (W / V) H ２ O ２Dissolve in 100 mL of (A) (prepared immediately before use and stored on ice) (C) Titanium Reagent (Nacalai Tesque) (D) Enzyme solution: Dilute the enzyme supplies to 0.35-1.35 mL with previously ice-cooled (A) (Procedure) 1. Place 0.25 mL of (B) in a test tube and preheat at 25°C for approximately 5 minutes. 2. Add 0.25 mL of (D) and mix gently. 3. After reacting for 5 minutes at 25°C, add 2.5 mL of (C) to stop the reaction, and measure the absorbance at 410 nm using water as a control (OD ｔｅｓｔ 4. For the blind test, after 5 minutes of reaction, first add 0.25 mL of (B) to 2.5 mL of (C) and mix, then add 0.25 mL of (D) (OD ｂｌａｎｋ )

[0059] The formula for calculating CAO activity is shown below. F: extinction coefficient of the titanium color product produced by 0.1 mM hydrogen peroxide (F is determined for each lot using hydrogen peroxide of known concentration. It is usually around 0.7)

[0060] The formula for calculating the CAO residual rate is shown below: CAO residual rate (%)=CAO activity value (U / mL) of reagent stored at 35°C for 1 week / CAO activity value (U / mL) of reagent stored at 4°C (Equation 2).

[0061] Table 1 shows the results of measuring catalase activity when imidazolidinyl urea was added to the first reagent of the creatinine measurement reagent.

[0062] When imidazolidinyl urea was not added, it was confirmed that the enzyme residual rate significantly decreased after storage at 35°C for one week (35°C, one week storage: 12.2 U / mL). On the other hand, the addition of imidazolidinyl urea resulted in an increase in the CAO residual rate depending on the imidazolidinyl urea concentration. Furthermore, it was found that the addition of imidazolidinyl urea stabilized CAO activity even in samples stored at 4°C (4°C, no addition: 88.5 U / mL → 4°C, 0.16 mM addition: 121.9 U / mL). From these results, it was found that the inclusion of imidazolidinyl urea in the first reagent (solution) stabilizes catalase activity.

[0063] Example 2: Confirmation of catalase activity value by addition of diazolidinyl urea Using the method for measuring catalase activity described above, catalase activity was measured when diazolidinyl urea was added to the first reagent of the creatinine measurement reagent.

[0064] Diazolidinyl urea was added to the first reagent of the creatinine measurement reagent at a concentration of 0.22 to 2.70 mM. As a comparative example, a first reagent without diazolidinyl urea was also prepared, and catalase activity was measured. The measurement results are shown in Table 2.

[0065] When diazolidinyl urea was not added, the residual enzyme rate was significantly reduced (10.28 U / mL) after storage at 35°C for one week. On the other hand, the addition of diazolidinyl urea resulted in an increase in the residual CAO rate in a diazolidinyl urea concentration-dependent manner. Furthermore, it was found that the addition of diazolidinyl urea improved stability even in samples stored at 4°C (4°C, no addition: 90.02 U / mL → 4°C, 0.22 mM addition → 115.1 U / mL). These results demonstrate that the inclusion of diazolidinyl urea in the first reagent (solution) stabilizes catalase activity.

[0066] Example 3: Confirmation of catalase activity value by addition of DMDM ​​hydantoin Using the method for measuring catalase activity described above, catalase activity was measured when DMDM ​​hydantoin was added to the first reagent of the creatinine measurement reagent.

[0067] DMDM hydantoin was added to the first reagent of the creatinine measurement reagent at a concentration of 0.33 to 3.99 mM. As a comparative example, a first reagent without DMDM ​​hydantoin was also prepared, and catalase activity was measured. The measurement results are shown in Table 3.

[0068] When DMDM ​​hydantoin was not added, it was confirmed that the residual rate of the enzyme was significantly reduced (10.28 U / mL) after storage at 35°C for one week. On the other hand, the addition of DMDM ​​hydantoin resulted in an increase in the residual rate of CAO. Furthermore, it was found that the addition of DMDM ​​hydantoin improved stability even in samples stored at 4°C (4°C, no addition: 90.92 U / mL → 4°C, 0.33 mM addition → 115.1 U / mL). These results confirmed that the addition of DMDM ​​hydantoin to the first reagent (solution) stabilizes catalase activity.

[0069] The stabilization method, the method for measuring a biological component, and the reagent or kit for measuring a biological component of the present invention can be used in fields such as in vitro diagnostic pharmaceuticals, and will make a great contribution to the industrial world.

Claims

1. A method for stabilizing a hemoprotein, comprising the step of allowing a hydantoin derivative to coexist.

2. The method according to claim 1, wherein the hydantoin derivative is a compound represented by the following formula (1): (In the formula, R1 to R3 are independently hydrogen or a substituent selected from the group consisting of an alkyl group, a hydroxyalkyl group, an amino group, a hydroxycarbonylamino group, a formylamino group, an acylamino group, a carbamoyl group, a carboxyl group, an aldehyde group, an acyl group, a hydroxyl group, a sulfone group, and a phenyl group, or a residue of a compound having at least one of the above-mentioned substituents; R2 and R3 may together form an oxo group or a ring; and R1, R2, and R3 each represent a group represented by formula (2): may be linked to R1, R2, and R3 of a compound represented by the formula:

3. The hydantoin derivative is N,N''-methylenebis[N'-[3-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]urea] (imidazolidinyl urea), N,N'-bis(hydroxymethyl)-N-[1,3-bis(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]urea (diazolidinyl urea), 1,3-bis(hydroxymethyl)-5,5-dimethylhydantoin (DMD 2. The method of claim 1, wherein the hydroxymethyl-1,2-dimethyl-2,4-dione is selected from the group consisting of 1,3-bis(hydroxymethyl)imidazolidine-2,4,5-trione, 1-(hydroxymethyl)-3-(2,3-dihydroxypropyl)-5,5-dimethylhydantoin, and 1,3-bis(hydroxymethyl)-1,3-diazaspiro[4.6]undecane-2,4-dione.

4. The method according to claim 2, wherein in formula (1), R1 is hydrogen or a hydroxyalkyl group.

5. The method according to claim 2, wherein in said formula (1), R2 is an alkyl group or an amino group, or a residue of a compound having an alkyl group and an amino group or an amide group.

6. The method according to claim 2, wherein in formula (1), R3 is hydrogen or an alkyl group.

7. The hydantoin derivative is represented by the formula (1-1) The method according to claim 1, wherein the imidazolidinyl urea is represented by the formula:

8. The hydantoin derivative is represented by the formula (1-2): The method according to claim 1, wherein the diazolidinyl urea is represented by the formula:

9. The hydantoin derivative is represented by the formula (1-3): The method according to claim 1, wherein the DMDM ​​hydantoin is represented by the formula:

10. The method according to claim 1, wherein the hydantoin derivative is contained in a reagent containing a hemoprotein at a concentration of 0.16 mM or more.

11. The method of claim 10, wherein the hemoprotein is catalase.

12. The method according to claim 11, wherein the concentration of catalase in the reagent is 10 to 500 KU / L.

13. The method according to claim 10, wherein the reagent is used to measure a biological component.

14. The method according to claim 13, wherein the biological component is at least one selected from the group consisting of creatinine, triglyceride, inorganic phosphorus, creatine, cholesterol ester, sialic acid, α-amylase, GOT, GPT, guarase, and phospholipids.

15. The method according to claim 13 or 14, wherein the biological component is creatinine.

16. A method for colorimetrically measuring a biological component by reacting a sample containing the biological component to be measured or thought to contain the biological component to be measured with at least one type of enzyme and then carrying out a color reaction, characterized in that a hemoprotein and a hydantoin derivative are allowed to coexist in the measurement reagent.

17. A reagent or kit for measuring a biological component, comprising at least the following (a) to (f) as components: (a) a hydantoin derivative, (b) a catalase, (c) a peroxidase, (d) one or more enzymes other than catalase and peroxidase, (e) a buffer, and (f) a color developer.

18. A reagent or kit for measuring a biological component as described in claim 17, wherein the (d) one or more enzymes other than catalase and peroxidase are at least one selected from the group consisting of ascorbic acid oxidase, sarcosine oxidase, creatine amidinohydrolase and creatinine amide hydrolase.

19. The reagent or kit for measuring a biological component according to claim 17, wherein the (e) buffer is at least one selected from the group consisting of Tris buffer, citrate buffer, borate buffer, phosphate buffer, MES, Bis-Tris, ADA, ACES, BES, PIPES, MOPS, TES, HEPES, Tricine, Bicine, POPSO, TAPS, CHES and CAPS.

20. The color former (f) is N-ethyl-N-(3-sulfopropyl)-m-anisidine, aniline, N,N-dimethylaniline, N,N-diethylaniline, N,N-diethyl-m-toludine, N,N-dimethyl-m-anisidine, N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine, N-ethyl-N-(β-hydroxyethyl)-m-toluidine, N-ethyl-N-(2-hydroxy-3-sulfoethyl)-m-toluidine, N-ethyl-N-sulfopropyl The reagent or kit for measuring a biological component according to claim 17, which is any one of p-m-toluidine, N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine, phenol, p-chlorophenol, 2,4-dichlorophenol, 2,4-dibromophenol, and 2,3,4-trichlorophenol.