Method for stabilizing heme protein by buffer agent

Abstract

To provide a method for stabilizing a heme protein suitable for use in biochemical and analytical chemistry fields, including clinical diagnostic applications.SOLUTION: The present invention provides a method for stabilizing a heme protein, which involves selecting a buffer agent having a -SO3H group, without employing a protein with a stabilizing effect.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for stabilizing hemoproteins. More specifically, it relates to a method for stabilizing catalase and peroxidase, which are hemoproteins, using a specific buffer.

Background Art

[0002] Conventionally, in clinical diagnosis, measurement of biological components by enzymatic methods has been carried out. In particular, a method using an oxidase - peroxidase - redox chromogenic reagent system, that is, a method in which a substance to be measured in a sample is subjected to an enzymatic reaction to generate hydrogen peroxide, and this is reacted with a chromogenic agent in the presence of peroxidase for colorimetric determination, is widely used. In order to make the composition containing the enzyme used in this method usable for a long period of time, it is of course important to stably maintain the enzyme activity.

[0003] Patent Document 1 describes a method for suppressing inactivation by an organic solvent by adding a protein folding factor to an organic solvent - containing solution containing a protein.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Patent Document 1 has found a method for suppressing inactivation of proteins by organic solvents, and is limited to a stabilization method under limited conditions. On the other hand, the present inventors have found that hemoproteins such as catalase and peroxidase are liable to decompose and denature in an aqueous solution and deactivate due to long - term storage. An object of the present invention is to provide a method for improving the stability of hemoproteins such as catalase in an aqueous solution. [Means for Solving the Problems]

[0006] As a result of intensive studies to solve the above problems, the present inventor has found that a hemoprotein can be stabilized by using a buffer having a -SO 3 H group, and has thus completed the present invention. That is, the present invention is configured as follows. (Item 1) -SO 3 A method for stabilizing a hemoprotein, characterized by using a buffer having a -SO (Item 2) The method according to Item 1, wherein the hemoprotein is catalase or peroxidase. (Item 3) The above-mentioned -SO 3The buffer having an H group is at least one solution selected from the group consisting of 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 3-morpholinopropanesulfonic acid (MOPS), N-tris(hydroxymethyl)methyl-2-aminomethanesulfonic acid (TES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (EPPS), 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPSO), 2-morpholinoethanesulfonic acid (MES), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) (POPSO), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) and N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPSO), the method according to claim 1 or 2. (Item 4) The method according to any one of claims 1 to 3, wherein the concentration of the buffer is 1 mmol / L to 1000 mmol / L. (Item 5) The buffer having an action of stabilizing the activity of catalase is at least one selected from the group consisting of MOPS, TES and HEPES, the method according to any one of claims 1 to 4. (Item 6) The buffer having an action of stabilizing the activity of peroxidase is at least one selected from the group consisting of MOPS and TES, the method according to any one of claims 1 to 5. (Item 7) ―SO 3A reagent kit for measuring biological components, characterized by coexisting a buffer having an H group and a heme protein, and containing at least the following components (a) to (d). (a) Catalase (b) Peroxidase (c) A redox chromogenic reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color (d) -SO 3 A buffer having an H group (Item 8) The method according to item 7, 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, guaase, and phospholipid. [Effect of the Invention]

[0007] According to the present invention, by using a specific buffer, a method for stabilizing a heme protein can be provided. [Modes for Carrying Out the Invention]

[0008] Hereinafter, the present invention will be described in more detail while showing 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, "~" in this specification means "above and below". For example, if "X~Y" is described in the specification, it means "X or more and Y or less". In addition, "and / or" in this specification means either one or both. Also, in this specification, it should be understood that the singular expression includes the concept of its plural form unless otherwise stated.

[0009] In addition, the enzyme to be stabilized in the present invention is not particularly limited in terms of its intended use, concentration, form of existence, etc. For example, as long as it is present in a composition that uses the enzyme as a constituent and utilizes the function of the enzyme, it can be exemplified by analytical reagents for molecular biology applications, analytical reagents for biochemical applications, in vitro diagnostic agents, liquid in vitro diagnostic agents, dry-type in vitro diagnostic agents processed into chip or slit shapes, enzyme sensors, enzyme electrodes, pharmaceutical compositions, etc. Manufacturing techniques and utilization techniques for these compositions have already been established in their respective industrial fields. Therefore, by applying the knowledge thereof to the present invention, the enzyme present in various compositions can be stabilized, and the mode thereof is not particularly limited.

[0010] (Method for stabilizing hemoprotein with buffer) The method of the present invention is to stabilize hemoprotein by using a buffer having a -SO 3 H group.

[0011] (Buffer) The buffer component used in the present invention desirably has a -SO 3 H group. There are no particular limitations on its usage amount, set pH, form of addition, etc. All of these can be obtained as commercially available products. For the stabilization of hemoprotein according to the present invention, it is considered that the hydrogen atom of the buffer forms a hydrogen bond with the non-bonding electron pair of the hemoprotein, prevents the denaturation of the hemoprotein, and maintains the reactivity between the enzyme and the substrate.

[0012] -SO 3Examples of buffers having an -SO₃H group include solutions such as 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 3-morpholinopropanesulfonic acid (MOPS), N-tris(hydroxymethyl)methyl-2-aminomethanesulfonic acid (TES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (EPPS), 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPSO), 2-morpholinoethanesulfonic acid (MES), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) (POPSO), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), and N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPSO).

[0013] The above, -SO₃ 3 Examples of solvents for buffers having an -SO₃H group include purified water, distilled water, RO water, ion-exchanged water, and Eli water.

[0014] In the present invention, -SO₃ 3 The type and concentration of the buffer having an -SO₃H group are not particularly limited. Preferably, the concentration in the reagent is adjusted to be 0 to 1000 mmol / L, preferably 5 to 100 mmol / L, more preferably 10 to 75 mmol / L, and even more preferably 20 to 50 mmol / L.

[0015] (Hemoprotein) A heme protein is a protein that contains heme. Heme means a complex composed 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 heme proteins include hemoglobin, which is an oxygen carrier, cytochromes involved in the electron transport system, catalase with enzyme activity, peroxidase, and the like. A heme protein may or may not have enzyme activity, but it preferably has enzyme activity. Examples of heme proteins with enzyme activity include catalase, peroxidase, and the like.

[0016] (Catalase) Catalase (EC 1.11.1.6) is a hemoprotein having protoheme and is an enzyme that catalyzes the reaction of decomposing hydrogen peroxide. As long as the effects of the present invention are exhibited, it is not particularly limited, and catalase derived from any microorganism (fungi, bacteria, archaea, etc.) can be used. For example, the catalase used in the present invention belongs to the genus Podospora, Neurospora, Cladosporium, Emericella, Pleurotus, Deinococcus, Escherichia, Salmonella, Pseudomonas, Bacillus, Mycobacterium, Botryotinia, Claviceps, Aspergillus, Ajellomyces, Agrobacterium, Sinorhizobium, Mesorhizobium, Nosema, Xanthomonas, Corynebacterium, or Arthrobacter. It can be a catalase derived from a microorganism, but is not limited thereto. From the viewpoint of more easily obtaining the effects of the present invention, it is preferable to use a catalase derived from a microorganism belonging to the genus Corynebacterium or the genus Arthrobacter.

[0017] The catalase used in the present invention may be catalase of any size, for example, catalase having a molecular weight (Mw) of about 50,000 to 90,000. In one embodiment, the catalase used in the present invention may be catalase having a molecular weight of less than 75,000, may also be catalase having a molecular weight of 65,000 or less, and further may be catalase having a molecular weight of 60,000 or less. By using catalase with such a relatively small molecular weight, it is expected to exhibit an even higher effect.

[0018] The catalase used in the present invention preferably has an optimum pH of pH 6.5 to 9.0, more preferably pH 7.2 to 8.5.

[0019] For the catalase used in the method of the present invention, only one type of catalase may be used, or two or more types of catalase may be mixed and used. Further, when two or more types of catalase are used, they may be catalase derived from the same type of microorganism or catalase derived from different types of microorganisms.

[0020] These microorganism-derived catalases are commercially available in various types, and commercially available products can be preferably used. Further, it can also be obtained by culturing microorganisms by a conventional method well known to those skilled in the art and purifying it from the culture.

[0021] (Peroxidase) As the peroxidase used in the present invention, any type of enzyme may be used as long as it is an enzyme that catalyzes the reaction between hydrogen peroxide and a redox chromogenic reagent. Examples include peroxidase derived from plants, bacteria, and basidiomycetes.

[0022] Among these, peroxidase derived from Japanese horseradish, rice, and soybean is preferable, and peroxidase derived from Japanese horseradish is more preferable, for reasons such as purity, ease of acquisition, and price.

[0023] As commercially available products, PEO-131 (manufactured by Toyobo), PEO-301 (manufactured by Toyobo), PEO-302 (manufactured by Toyobo), etc. are preferably used.

[0024] The peroxidase used in the present invention may be peroxidase of any size, for example, peroxidase having a molecular weight (Mw) of about 30,000 to 50,000. In one embodiment, the peroxidase used in the present invention preferably has a molecular weight of 40,000 to 45,000.

[0025] The peroxidase used in the present invention preferably has an optimum pH of pH 5.5 to 9.0, more preferably pH 6.0 to 7.0.

[0026] For the peroxidase used in the method of the present invention, only one type of peroxidase may be used, or two or more types of peroxidases may be mixed and used. Further, when two or more types of peroxidases are used, they may be peroxidases derived from the same type of microorganism or peroxidases derived from different types of microorganisms.

[0027] (Biological component measurement reagent) The biological component measurement reagent includes analytical reagents for biochemical use, in vitro diagnostic agents, liquid in vitro diagnostic agents, etc. Further, in a broad sense, it also includes dry-type in vitro diagnostic agents processed into chip shape or slit shape, enzyme sensors, enzyme electrodes, etc.

[0028] The biological component measurement reagent of the present invention is not particularly limited as long as it contains the following components (1) to (4). As described above, it can take various forms, and those skilled in the art can appropriately set the constitution such as the reagent composition based on the description of this specification. The biological component measurement reagent kit may further include a specimen sampler, an instruction manual, etc. in addition to the bottle containing the first reagent and the bottle containing the second reagent. (1) Peroxidase (2) Catalase (3) Redox chromogenic reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color (4)-SO 3 Buffer having an H group

[0029] As the biological component measurement reagent, a CRE measurement reagent is preferred. The CRE measurement reagent is not particularly limited as long as it can quantify creatinine, but a reagent containing creatinine amidohydrolase, creatin amidohydrolase, sarcosine oxidase, and peroxidase is preferred. Such a CRE measurement reagent can quantify the CRE concentration by a peroxidase-chromogenic agent system by designing a so-called conjugate reaction in which creatine produced by the reaction of creatinine amidohydrolase using creatinine as a substrate is further reacted with creatin amidohydrolase to produce sarcosine, and further hydrogen peroxide is produced from sarcosine using sarcosine oxidase.

[0030] The UA measurement reagent is not particularly limited as long as it can quantify uric acid, but a reagent containing uricase and peroxidase is preferred. Such a UA measurement reagent can quantify the UA concentration by a peroxidase-chromogenic agent system by the hydrogen peroxide produced by the reaction of uricase using uric acid as a substrate.

[0031] The TG measurement reagent is not particularly limited as long as it can quantify triglyceride, but a reagent containing lipoprotein lipase, glycerol kinase, glycerol 3-phosphate oxidase, and peroxidase is preferred. Such a TG measurement reagent can quantify the TG concentration by a peroxidase-chromogenic agent system by producing hydrogen peroxide using lipoprotein lipase using triglyceride as a substrate and glycerol kinase and glycerol 3-phosphate oxidase as conjugate enzymes.

[0032] The LDL and HDL measurement reagents are not particularly limited as long as they can quantify LDL cholesterol and HDL cholesterol, but reagents containing cholesterol esterase and cholesterol oxidase are preferred. Such LDL and HDL measurement reagents can quantify LDL and HDL by the peroxidase - chromogenic agent system by allowing cholesterol esterase and cholesterol oxidase to act to produce hydrogen peroxide.

[0033] As a means for implementing the above method, it is preferably a reagent (or kit) configured to be applicable to a general - purpose automatic analyzer (for example, Hitachi 7170 type automatic analyzer). As such a form, for example, a reagent (or kit) composed of a combination of a dry preparation produced by means such as freeze - drying and a dissolution solution can be mentioned. Preferably, it is a liquid reagent, for example, a liquid reagent in which the reagent is divided into two packages (hereinafter, also referred to as a liquid reagent of a two - reagent system). In this method, first, the first type of reagent (hereinafter, also referred to as the first reagent) is added to the sample and allowed to react for a certain period of time, then the second type of reagent (hereinafter, also referred to as the second reagent) is further added and allowed to react, and the target component can be quantified by measuring the change in absorbance during this period.

[0034] (Redox chromogenic reagent) As the redox chromogenic reagent used in the present invention, any type of dye may be used as long as it reacts with hydrogen peroxide to develop color. For example, hydrogen donors, couplers, leuco forms, tetrazolium salts, etc. can be mentioned. There are no particular limitations on its usage amount, form of addition, etc. These can all be obtained as commercially available products.

[0035] A typical example using a hydrogen donor and a coupler is the Trinder method in which the hydrogen donor and the coupler are oxidatively condensed with hydrogen peroxide in the presence of peroxidase to form a dye. As hydrogen donors used in the Trinder method and the like, phenol, phenol derivatives, aniline derivatives, naphthol, naphthol derivatives, naphthylamine, naphthylamine derivatives and the like are known. For example, N-ethyl-N-sulfopropyl-3-methoxyaniline, N-ethyl-N-sulfopropyl aniline, 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-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-sulfopropyl aniline, N-(2-hydroxy-3-sulfopropyl)-2,5-dimethylaniline, N-ethyl-N-(3-methylphenyl)-N'-succinylethylenediamine, N-ethyl-N-(3-methylphenyl)-N'-acetyethylenediamine and the like can be mentioned. Further, these hydrogen donors can be used in combination with a coupler. As the coupler, 4-aminoantipyrine (4AA), aminoantipyrine derivatives, vanillinediamine sulfonic acid, methylbenzothiazolinone hydrazone (MBTH), sulfonated methylbenzothiazolinone hydrazone (SMBTH) and the like are known.

[0036] Examples of the leuco form include triphenylmethane derivatives, phenothiazine derivatives, diphenylamine derivatives, etc. Specifically, 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), 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine salt (DA67), etc. are included.

[0037] Examples of the tetrazolium salt 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, 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-diyl]-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, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium salt, etc.

[0038] In the present invention, preservatives, salts, enzyme stabilizers, chromogen stabilizers, etc. may be added within a range that does not affect the reaction. Their usage amounts, forms of addition, etc. are not particularly limited. Any of these can be obtained as commercially available products. Examples of preservatives include azides, chelating agents, antibiotics, antibacterial agents, etc. Examples of chelating agents include ethylenediaminetetraacetic acid and its salts. Examples of antibiotics include gentamicin, kanamycin, chloramphenicol, etc. Examples of antibacterial agents include methylisothiazolinone, imidazolidinyl urea, etc. Examples of salts include sodium chloride, potassium chloride, aluminum chloride, etc. Examples of enzyme stabilizers include sucrose, trehalose, cyclodextrin, gluconates, amino acids, etc. Examples of chromogen stabilizers include chelating agents such as ethylenediaminetetraacetic acid and its salts, cyclodextrin, etc.

Example

[0039] Hereinafter, the present invention will be specifically described by way of examples. Note that the present invention is not particularly limited by the examples.

[0040] <Example 1: Catalase Activity Measurement> Catalase was dissolved in various buffer solutions, and the stability of catalase activity was evaluated. The types of various buffer solutions used were Bis-Tris, MOPS, TES, HEPES, and Bicine, and the concentration was 50 mmol / L. Also, the concentration of catalase was 120.0 U / mL.

[0041] For the measurement, two types of the above reagents were used: the reagent stored at 4°C and the reagent stored at 35°C for 1 week.

[0042] (Measurement Method) Catalase (CAO) (Principle)

Chemical Formula

[0043] (Reagents) (A) 10 mmol / L phosphate buffer, pH 7.0 (B) H 2 O 2 Solution: Dissolve 16 mmol / L [0.182 mL of 30% (W / V) H 2 O 2 in 100 mL of (A) (Prepare as needed and store at ice-cold temperature). (C) Titanium reagent (manufactured by Nacalai Tesque) (D) Enzyme solution: Dilute the enzyme preparation with pre-cooled (A) to 0.35 - 1.35 mL.

[0044] (Procedure) 1. Take 0.25 mL of (B) in a test tube and pre-warm at 25 °C for about 5 minutes. 2. Add 0.25 mL of (D) and mix gently. 3. After reacting exactly 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 the 5-minute 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 )

[0045] The calculation formula for CAO activity is described below.

Equation

[0046] The calculation formula for CAO residual rate is described below.

Equation

[0047] Peroxidase (PEO) (Principle) Extract the generated purpurogallin with ether and measure it by the change in absorbance at 420 nm. (Definition) The amount of enzyme that generates 1.0 mg of purpurogallin in 20 seconds under the following conditions is defined as 1 purpurogallin unit (U).

[0048] (Reagents) (A) 5% (W / V) aqueous pyrogallol solution (prepared as needed) (B) 0.147 M H 2 O 2 aqueous solution (1.67 mL of 30% (W / V) H 2 O 2 solution is diluted with distilled water to 100 mL) (C) 0.1 mol / L phosphate buffer pH 6.0 (for reaction mixture and enzyme dilution) (D) 2.0 N H 2 SO 4 solution (E) Enzyme solution: Dissolve the enzyme standard in pre-cooled (C) in advance, dilute it to 3.0 - 6.0 Purpurogallin U / mL with (C), and store it in ice-cooled condition.

[0049] (Procedure) 1. Prepare the following reaction mixture in a test tube and pre-warm it at 20 °C for about 5 minutes. 14.0 mL distilled water 2.0 mL aqueous pyrogallol solution (A) 1.0 mL H 2 O 2 aqueous solution (B) 2.0 mL phosphate buffer (C) 2. Add 1.0 mL of (E) to start the reaction. 3. After reacting accurately for 20 seconds at 20 °C, add 1.0 mL of (D) to stop the reaction. Extract the generated purpurtogallin from the reaction mixture after stopping with 15 mL of ether. Repeat this operation 5 times, combine the extracts, and add more ether to make the total volume 100 mL. Measure the absorbance at 420 nm for this solution (OD test ). 4. For the blind test, after leaving the reaction mixture at 20°C for 20 seconds, add (D) 1.0 mL and mix, then add (E) 1.0 mL to prepare. Perform ether extraction on this solution in the same manner as above and measure the absorbance (OD blank ).

[0050] The calculation formula for PEO activity is described below.

Number

[0051] The calculation formula for PEO residual rate is described below.

Number

[0052] Table 1 shows the measurement results of catalase activity when catalase was dissolved in various buffer solutions.

Table 1

[0053] From these measurement results, it was found that the catalase activity can be stabilized by using MOPS, TES, and HEPES. Also, when measurements were carried out under the conditions of pH 6.0 and pH 7.7 using MOPS, the result that the catalase activity was stabilized under both conditions was obtained. That is, it was found that it is the stabilizing action of the buffer solution, not the stabilization by pH.

[0054] <Example 2: Peroxidase Activity Measurement> Peroxidase was dissolved in various buffers, and the stability of peroxidase activity was evaluated. The types of various buffers used were Bis-Tris, MOPS, TES, HEPES, and Bicine, and the concentration was 50 mmol / L. Also, the concentration of peroxidase was 8.0 U / mL.

[0055] For the measurement, two types of the above reagents were used: the reagent stored at 4°C and the reagent stored at 35°C for one week.

[0056] Table 2 shows the measurement results of peroxidase activity when peroxidase was dissolved in various buffers.

Table 2

[0057] From these measurement results, it was found that the peroxidase activity can be stabilized by using MOPS and TES. Also, when the measurement was carried out under the conditions of pH 6.0 and pH 7.7 using MOPS, the peroxidase activity was stabilized under both conditions. That is, it was found that it is the stabilizing action of the buffer, not the stabilization by pH.

Industrial Applicability

[0058] The measurement method of the present invention can be used in application fields such as pharmaceuticals for in vitro diagnosis, and it is of great contribution to the industrial world.

Claims

Claim 1 -SO ３ A method for stabilizing a hemoprotein, characterized by using a buffer having an -SH group. Claim 2 The method according to claim 1, wherein the heme protein is catalase or peroxidase. Claim 3 The aforesaid, -SO ３ The method according to claim 1, wherein the buffer having an -SH group is at least one solution selected from the group consisting of 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 3-morpholinopropanesulfonic acid (MOPS), N-tris(hydroxymethyl)methyl-2-aminomethanesulfonic acid (TES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (EPPS), 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPSO), 2-morpholinoethanesulfonic acid (MES), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) (POPSO), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) and N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPSO). Claim 4 The method according to claim 1 or 2, wherein the concentration of the buffer is 1 mmol / L to 1000 mmol / L. Claim 5 The method according to claim 4, wherein the buffer having an effect of stabilizing the activity of catalase is at least one selected from the group consisting of MOPS, TES, and HEPES. Claim 6 The method according to claim 4, wherein the buffer having an effect of stabilizing the activity of peroxidase is at least one selected from the group consisting of MOPS and TES. Claim 7 -SO ３ A reagent kit for measuring biological components, characterized by coexisting a buffer having an -SH group and a hemoprotein, and containing at least the following components (a) to (d). (a) Catalase (b) Peroxidase (c) A redox chromogenic reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color (d)-SO ３ Buffer with -H group Claim 8 The method according to claim 7, 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, guaase, and phospholipid.

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