JP2004173519A - Method for stabilizing d-amino acid oxidase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for stabilizing a D-amino acid oxidase and to provide a reagent for assaying homocysteine having high stability by utilizing the method. <P>SOLUTION: The method for stabilizing the D-amino acid oxidase comprises a step of making the D-amino acid oxidase coexist with a dithiodinicotinic acid in a liquid. Furthermore, an anionic surfactant is preferably contained in the liquid. A kit for assaying the homocysteine comprising the reagent containing a thiol compound, a homocysteine methyltransferase and D-methionine methylsulfonium and preferably further a reagent for assaying a D-amino acid containing the D-amino acid oxidase, the dithiodinicotinic acid and an anionic surfactant is provided. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for stabilizing D-amino acid oxidase, and a reagent for measuring homocysteine using this method.
[0002]
[Prior art]
To biochemically quantify homocysteine, homocysteine in a sample treated with a reducing agent is contacted with adenosine and S-adenosyl-L-homocysteine hydrolase, and the amount of adenosine in the remaining mixture is evaluated. There is a known method (see Patent Document 1). However, in the presence of a reducing agent such as a thiol compound that is used for reduction treatment, which is essential for measuring total homocysteine in blood, the generated hydrogen peroxide is converted into a measurement system using a commonly used oxidizing color former. There is a problem that it cannot be guided. This is because the thiol compound significantly inhibits the color development of the oxidizing color former. For this reason, this method cannot be applied to a general-purpose automatic analyzer.
[0003]
In order to solve this problem, a method using an SH reagent in the post-reduction process has been reported (see Patent Document 2). In this method, the remaining homocysteine auxiliary substrate, the produced homocysteine converting enzyme product, or the enzyme reaction product thereof is oxidized in the presence of the SH reagent to produce hydrogen peroxide, and at the same time, the SH reagent is used. It can be used to block the action of excess thiol compounds. However, this SH reagent is highly reactive and may cause significant deterioration in the stability of coexisting enzymes. For example, in a method that uses D-amino acid oxidase to lead to a measurement system using an oxidative color former, the addition of SH reagent makes D-amino acid oxidase unstable, causing the storage stability of the measurement reagent itself to deteriorate. It was.
[0004]
[Patent Document 1]
Japanese Patent No. 2870704 [Patent Document 2]
International Publication No. 02/02802 Pamphlet [0005]
[Problems to be solved by the invention]
As a stabilizer for D-amino acid oxidase, benzoic acid is well known, but at the same time, it has a problem of showing a strong inhibitory action on the enzyme. An object of the present invention is to provide a method for stabilizing D-amino acid oxidase and a highly stable reagent for measuring homocysteine using this method.
[0006]
[Means for Solving the Problems]
As a result of repeated studies to achieve the above object, it has been found that dithiodinicotinic acid has a D-amino acid oxidase stabilizing action and an enzyme inhibiting action is negligibly small. Furthermore, it has also been found that by using an anionic surfactant, it is possible to stabilize D-amino acid oxidase and to prevent precipitation of insoluble substances that are considered to be generated by denaturation.
[0007]
The present invention provides a method for stabilizing D-amino acid oxidase, which comprises the step of allowing D-amino acid oxidase and dithiodinicotinic acid to coexist in a liquid.
[0008]
In a preferred embodiment, the liquid contains an anionic surfactant.
[0009]
In a more preferred embodiment, the anionic surfactant comprises polyoxyethylene alkylalkyl ether sulfate, polyoxyethylene alkylphenyl ether sulfate, polyoxyethylene dialkyl allyl ether sulfate, polyoxyethylene alkenyl allyl ether sulfate, and their It is at least one compound selected from the group consisting of salts.
[0010]
In another preferred embodiment, the D-amino acid oxidase is a porcine kidney D-amino acid oxidase.
[0011]
The present invention also provides a D-amino acid oxidase-containing solution containing dithiodinicotinic acid.
[0012]
In a preferred embodiment, the D-amino acid oxidase-containing solution further contains an anionic surfactant.
[0013]
The present invention also provides a reagent for measuring D-amino acid comprising any one of the above-mentioned D-amino acid oxidase-containing liquids.
[0014]
The present invention further provides a kit for measuring homocysteine, comprising a reagent containing a thiol compound, homocysteine methyltransferase, and D-methionine methylsulfonium, and the above-mentioned reagent for measuring D-amino acid.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
Dithiodinicotinic acid (6,6'-dithiodinicotinic acid) is a D-amino acid oxidase [EC in a D-amino acid oxidase-containing solution under all circumstances from the case of purifying D-amino acid oxidase to the case of using it. 1.4.3.3] is stabilized. For example, when D-amino acid oxidase is purified by an operation usually used in the presence of dithiodinicotinic acid, a purified enzyme can be obtained while preventing a decrease in enzyme activity. In addition, for example, inactivation in a freeze-drying step of a D-amino acid oxidase-containing preparation can be prevented. Further, for example, when an SH reagent such as N-ethylmaleimide coexists like a D-amino acid oxidase-containing liquid for homocysteine measurement, the stability of D-amino acid oxidase can be achieved by adding dithiodinicotinic acid to this liquid. Sex can be improved dramatically.
[0016]
The concentration of dithiodinicotinic acid is not particularly limited as long as it is an effective concentration for stabilizing D-amino acid oxidase. For example, in the homocysteine measurement method to be described later, in the range of 0.001 to 100 mM, preferably 0.01 to 10 mM, more preferably 0.05 to 2 mM with respect to D-amino acid oxidase 0.1 to 100 U / ml. Can be used.
[0017]
An anionic surfactant not only stabilizes D-amino acid oxidase in a D-amino acid oxidase-containing solution, but also prevents the occurrence of turbidity that occurs when an SH reagent such as N-ethylmaleimide is contained in this solution. Can do. An example of the case in which the SH reagent is included includes the homocysteine measurement method described later.
[0018]
The anionic surfactant is at least selected from the group consisting of polyoxyethylene alkyl ether sulfuric acid, polyoxyethylene alkyl phenyl ether sulfuric acid, polyoxyethylene dialkyl allyl ether sulfuric acid, polyoxyethylene alkenyl allyl ether sulfuric acid, and salts thereof. One type is preferably used. One of these may be used alone, or two or more may be used. More specifically, polyoxyethylene alkyl ether sulfates [Example: Haitenol 18E (Daiichi Kogyo Seiyaku Co., Ltd.); Emar 20C and 20A, Lebenol WX (above, Kao Corporation)]], polyoxy Ethylene alkyl phenyl ether sulfuric acid [Example: Haitenol N-17 (Daiichi Kogyo Seiyaku Co., Ltd.); Emar NC, Lebenol WZ (above, Kao Co., Ltd.)], polyoxyethylene dialkyl allyl ether sulfuric acid [Example: Hytenol NE-15 (Daiichi Kogyo Seiyaku Co., Ltd.)], polyoxyethylene alkenyl allyl ether sulfate [Example: Hitenol NF-13 (Daiichi Kogyo Seiyaku Co., Ltd.)] and the like.
[0019]
The concentration of the anionic surfactant is not particularly limited as long as it is in a range effective for stabilizing D-amino acid oxidase and does not adversely affect homocysteine measurement. For example, in the homocysteine measurement method described later, it is preferable to use 0.001 to 5% (w / v), preferably 0.01 to 0.5% (w / v).
[0020]
As a typical example of using a D-amino acid oxidase-containing solution, a method for measuring homocysteine will be specifically described (see Patent Document 2).
[0021]
In the method for measuring homocysteine using D-amino acid oxidase, it is generated after a methyltransferase is allowed to act on homocysteine in a sample subjected to reduction treatment with a thiol compound in the presence of a methyl donor (hereinafter referred to as the first step). A D-amino acid oxidase is allowed to act on a D-amino acid derivative or D-amino acid analog in the presence of SH reagent and dithiodinicotinic acid, and the generated hydrogen peroxide is measured by an oxidizing color former (hereinafter referred to as the second step). ). In the second step, it is preferable to use an anionic surfactant.
[0022]
The test sample used in this method may be any sample that is considered to contain homocysteine. In addition to the reduced homocysteine, homocysteine exists in the form of oxidized homocysteine bound to other molecules by disulfide bonds such as protein-bound, homocysteine dimer, homocysteine-cysteine dimer. Either is acceptable. Examples include serum, plasma, blood, urine, and dilutions thereof.
[0023]
The thiol compound used in this method is not particularly limited, and examples thereof include dithiothreitol, mercaptoethanol, N-acetylcysteine, dithioerythritol, and thioglycolic acid. The concentration of the thiol compound may be any as long as it can convert oxidized homocysteine to reduced homocysteine, and is preferably 0.1 mM or more, more preferably 1 mM or more as a thiol group.
[0024]
Examples of the methyl donor include D-methionine methylsulfonium, S-adenosyl-D-methionine, D-ethionine methylsulfonium, and the like. Preferably, D-methionine methylsulfonium is used.
[0025]
Any methyltransferase may be used as long as it acts on D-methionine methylsulfonium and L-homocysteine and catalyzes the production of D-methionine and L-methionine. For example, homocysteine methyltransferase [ EC 2.1.1.10], 5-methyltetrahydrofolate-homocysteine S-methyltransferase [EC 2.1.1.13], 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase [EC 2.1.1.14]. Preferably homocysteine methyltransferase [EC 2.1.1.10.] Is used. The strain name of homocysteine methyltransferase is S-adenosyl-L-methionine: L-homocysteine S-methyltransferase, which is a substrate of L-homocysteine of methyl acceptor and S-adenosyl-L-methionine of methyl donor. And L-methionine and S-adenosyl-L-homocysteine (Enzyme Handbook, Asakura Shoten, 1982). S. K. Shapiro reports that this enzyme also utilizes S-methyl-L-methionine (L-methionine methylsulfonium), or S-adenosyl-D-methionine as a methyl donor (Biochim. Biophys. Acta). , 29, 405-409, 1958).
[0026]
The homocysteine methyltransferase used can be of any origin as long as it uses D-methionine methylsulfonium as the methyl donor. For example, enzymes derived from bacteria, yeast, rats and the like can be used.
[0027]
D-methionine is quantified using D-amino acid oxidase [EC 1.4.3.3]. Since D-methionine methylsulfonium, which is one of the D-amino acids, hardly becomes a substrate for D-amino acid oxidase, D-amino acid converting enzyme does not act on D-methionine methylsulfonium, or even if it acts, D- If the reactivity is sufficiently lower than that for methionine, the produced D-methionine is measured without removing D-methionine methylsulfonium remaining after the reaction with homocysteine methyltransferase outside the reaction system. Can do.
[0028]
The D-amino acid oxidase used can be of any origin. For example, enzymes derived from bacteria, invertebrates, vertebrates and the like can be used. Most preferred is an enzyme from porcine kidney.
[0029]
When D-amino acid oxidase is allowed to act on D-methionine, hydrogen peroxide is produced. This can be led to an oxidative color former usually used in the presence of an SH reagent, and colorimetrically determined. The generated hydrogen peroxide can cause a normal oxidizing color former to develop color with peroxidase. This coloring method is a known method generally used in the field of clinical chemistry. However, since the coloring of the thiol compound used in the first step is significantly hindered by the reducing action, it is essential to add an SH reagent that is a blocking agent for the thiol compound in the second step. Therefore, the addition of the SH reagent in the second step prevents the S-adenosyl-L-homocysteine hydrolase used in the first step from catalyzing a reverse reaction (hydrolysis reaction) in the second step, It has the effect of preventing coloration interference of the oxidative color former due to the thiol compound.
[0030]
As the SH reagent, as described in the Biochemical Dictionary (3rd edition, p.182, Tokyo Kagaku Dojin, 1998), an oxidizing agent such as an Elman reagent, a mercapto-forming agent such as p-methacrylic benzoic acid, and iodine. Examples thereof include alkylating agents such as acetic acid and N-ethylmaleimide. Preferably alkylating agents are used, more preferably maleimide compounds, most preferably N-ethylmaleimide.
[0031]
The concentration of the SH reagent in the second step may be any as long as it can block the thiol group of the thiol compound used for the reduction treatment of the sample to the extent that the quantification by the oxidizing color former is not hindered, preferably from 0.1 mM. It can be used in the range of 100 mM. More preferably, it is used at 1 mM to 30 mM, but in order to exert the effect of inhibiting S-adenosyl-L-homocysteine hydrolase, it is desirable to use an excessive amount of SH reagent compared to the thiol compound used. .
[0032]
As the oxidizing color former, various trinder reagents can be used in combination with the coupler reagent. This method is also referred to as the Trinder method and is commonly used in the field of clinical chemistry analysis and is not described in detail here, but preferably 4-aminoantipyrine as the coupler reagent and ADOS [N-ethyl-N as the tender reagent. -(2-hydroxy-3-sulfopropyl) -3-methoxyaniline], DAOS [N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline], HDAOS [N- ( 2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline], MAOS [N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline], TOOS [N-ethyl -N- (2-hydroxy-3-sulfopropyl) -3-methylaniline] and the like are used. Also, no coupler reagent is required, o-tolidine, o-dianisidine, DA-67 [10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine sodium, Wako Pure Chemical Industries, Ltd. Manufactured], TPM-PS [N, N, N ′, N ′, N ″, N ″ -hexa (3-sulfopropyl) -4,4 ′, 4 ″ -triaminotriphenylmethane 6 sodium salt, Dojindo Chemical Co., Ltd. A leuco-type color reagent such as “Laboratory Laboratory” can be used in the same manner, in particular, DA-67 and TPM-PS can be measured with higher sensitivity because they have a larger molar extinction coefficient than the above-mentioned Trinder reagent.
[0033]
The kit for measuring homocysteine according to the present invention comprises (1) a thiol compound for reducing homocysteine, a methyltransferase (preferably homocysteine methyltransferase), and a methyl donor (preferably D-methionine methylsulfonium). And (2) a D-amino acid measurement reagent containing D-amino acid oxidase and dithiodinicotinic acid for measuring the produced D-methionine. Preferably, the reagent for measuring D-amino acid (2) also contains an anionic surfactant. The reagent for measuring D-amino acid (2) preferably further contains an SH reagent (preferably N-ethylmaleimide), peroxidase, and an oxidizing color former. The oxidizing coloring reagent may be included in the reagent (1).
[0034]
【Example】
Example 1: Stabilizing effect of dithiodinicotinic acid (0.5 mM) on D-amino acid oxidase and homocysteine measurement reagent 150 mM 2-morpholinoethanesulfonic acid (pH 6.0), 20 mM N-ethylmaleimide, 4 U / mL Porcine kidney-derived D-amino acid oxidase, 5.5 U / mL peroxidase, 0.05% Triton X-100, and further, a test substance 6,6′-dithiodinicotinic acid (DTNA), A solution containing 0.5 mM of nicotinic acid (NA) or benzoic acid (BA) was prepared. The D-amino acid oxidase (DAO) activity was measured immediately after preparation and after storage at 7 ° C. for 3 days and 7 days, and the residual activity was calculated (FIG. 1). DAO activity was measured using a Hitachi 7170 automatic analyzer as follows. 180 μL of the first reagent containing 120 mM bicine (pH 8), 1 mM TOOS, and 1.5 mM FAD was added to 5 μL of the solution immediately after preparation or after storage, and reacted at 37 ° C. for about 5 minutes. Subsequently, 60 μL of a second reagent containing 120 mM bicine (pH 8), 3 mM 4-aminoantipyrine, 13.2 U / mL peroxidase, and 64 mM D-methionine was added and reacted at 37 ° C. for about 5 minutes. The change in absorbance (main wavelength 546 nm, subwavelength 700 nm) at measurement points 16 to 34 was determined.
[0035]
At the same time, these solutions were used as a second reagent for homocysteine (Hcy) measurement, and a sample containing 50 μM homocysteine (Hcy) was measured using a Hitachi 7170 automatic analyzer (FIG. 2). That is, to 15 μL of a sample containing 25 μM Hcy, 150 mM bicine, 100 U / L homocysteine methyltransferase, 5.6 mM dithiothreitol, 0.06 mM D-methionine methylsulfonium, 1 mM zinc bromide, and 0.3 mM DA-67 were added. 180 μL of the first reagent was added and reacted at 37 ° C. for about 5 minutes. Next, 120 μL of the second reagent was added thereto, and further reacted at 37 ° C. for about 5 minutes. The change in absorbance (main wavelength 660 nm, subwavelength 700 nm) at measurement points 16 to 34 was determined.
[0036]
FIG. 1 shows the relative activity after storage for 3 days and 7 days at 7 ° C., with the activity immediately after the preparation of the reagent as 100. In any liquid containing DTNA, NA, or BA, the activity of DAO was higher than that in the case of no addition.
[0037]
FIG. 2 shows the relative sensitivity when measured with a reagent after storage at 7 ° C. for 3 days and 7 days, with the sensitivity when measuring Hcy in a sample using a reagent immediately after preparation (no additive) as 100. Sensitivity was shown. The reagent to which DTNA was added retained 100% sensitivity even after storage for 3 days. On the other hand, BA was very poor in measurement sensitivity immediately after preparation due to its DAO inhibitory action. Moreover, although it did not reach DTNA, NA also had little decrease in measurement sensitivity.
[0038]
Example 2: DAO stabilization effect of DTNA and polyoxyethylene alkyl ether sulfate 150 mM N- (2-acetamido) iminodiacetic acid (pH 6.0), 23 mM N-ethylmaleimide, 4 U / mL Pig kidney-derived DAO Liquid A containing 5 U / mL peroxidase and 0.05% Triton X-100, Liquid B containing 0.05% Haitenol 18E instead of 0.05% Triton X-100 in Liquid A, and Liquid B Further, C solution containing 0.8 mM DTNA was prepared, and the DAO activity was measured. Next, after storage at 25 ° C. for 1 day and 9 days, DAO activity was measured again, and the residual activity was calculated (FIG. 3).
[0039]
In the liquid A, precipitates were observed during storage, but the liquid B and liquid C containing hytenol 18E were clear. Moreover, as shown in FIG. 3, the DAO stabilizing effect was observed slightly with Haitenol 18E than with Triton X-100. Furthermore, it was revealed that DAO is significantly stabilized by DTNA.
[0040]
Example 3: Stabilizing effect of DTNA (0.8 mM) on DAO and Hcy measurement reagents 150 mM 2-morpholinoethanesulfonic acid (pH 6.0), 23 mM N-ethylmaleimide, 4 U / mL porcine kidney-derived DAO, A solution containing 5 U / mL peroxidase and 0.05% hytenol 18E, and 0.8 mM DTNA, 1.3 mM NA, 0.8 mM 2,2′-dithiodibenzoic acid (DTBA), or A solution containing 0.1 mM BA was prepared. The DAO activity was measured immediately after preparation and after storage at 7 ° C. for 6 days, and the residual activity was calculated (FIG. 4). At the same time, using these solutions as the second reagent for Hcy measurement, a sample containing 50 μM Hcy was measured (FIG. 5).
[0041]
FIG. 4 shows the relative activity after storage for 6 days at 7 ° C., with DAO activity immediately after the preparation of the reagent as 100. In the reagent without additive, the residual activity decreased to about 10%, whereas in DTNA and BA, about 80%, and in NA and DTBA, about 60% of the activity remained, and a DAO stabilization effect was observed. .
[0042]
FIG. 5 shows the relative sensitivity when the Hcy measurement sensitivity immediately after the preparation of the additive-free reagent was set to 100, and the measurement was performed using the reagent after storage at 7 ° C. for 6 days. In the reagent without additive, the sensitivity decreased to 50% or less after 6 days storage at 7 ° C., whereas when DTNA was added, the sensitivity was maintained at 100%. On the other hand, in the case of NA, DTBA, and BA, because of their strong inhibitory action, the measurement sensitivity was about 40% with respect to the sensitivity of the additive-free reagent immediately after preparation.
[0043]
Example 4: Stabilizing effect of DTNA on lyophilized formulation A solution containing 200 mM citrate buffer (pH 5.6), 16 U / mL porcine kidney-derived DAO, 22 U / mL peroxidase, and 1% lactose, and A solution containing 1.6 mM of the test substance DTNA was prepared in this solution, and 1.5 mL each was freeze-dried. Each freeze-dried preparation was condensed with a solution containing 25 mM N-ethylmaleimide, and each DAO activity was measured. Further, a 50 mM phosphate buffer (pH 7.0) was used instead of the buffer used for the stock solution of the lyophilized preparation, and a 50 mM citrate buffer (pH 5) containing 25 mM N-ethylmaleimide was used instead of the lysate. The lyophilized preparation operated in the same manner as described above was also examined except that .6) was used (FIG. 6).
[0044]
As can be seen from FIG. 6, it was revealed that DTNA can prevent DAO inactivation in any lyophilized preparation.
[0045]
【The invention's effect】
According to the method of the present invention, D-amino acid oxidase can be stabilized. Therefore, a reagent or preparation containing D-amino acid oxidase having higher enzyme activity than conventional products can be obtained. Further, in measurement using D-amino acid oxidase, measurement with higher sensitivity is possible. Furthermore, the preservability of the reagent or formulation containing D-amino acid oxidase is also improved.
[Brief description of the drawings]
FIG. 1 is a graph showing the time course of residual DAO activity in a D-amino acid oxidase (DAO) -containing solution containing various additives (DTNA, NA, or BA).
FIG. 2 is a graph showing changes in sensitivity of Hcy measurement over time with a homocysteine (Hcy) measurement reagent containing various additives (DTNA, NA, or BA).
FIG. 3 is a graph showing residual DAO activity after storage for 1 day and 9 days in a D-amino acid oxidase (DAO) -containing solution containing a surfactant and / or DTNA.
FIG. 4 is a graph showing residual DAO activity after 6 days storage in a D-amino acid oxidase (DAO) -containing solution containing various additives (DTNA, NA, DTBA, or BA).
FIG. 5 is a graph showing the relative sensitivity at the time of Hcy measurement immediately after preparation of an Hcy measurement reagent containing various additives (DTNA, NA, DTBA, or BA) and after storage for 6 days.
FIG. 6 is a graph showing DAO relative activity in DAO lyophilized formulations containing DTNA.

Claims (8)

A method for stabilizing D-amino acid oxidase, comprising a step of allowing D-amino acid oxidase and dithiodinicotinic acid to coexist in a liquid. The method of claim 1, wherein the liquid comprises an anionic surfactant. The anionic surfactant is selected from the group consisting of polyoxyethylene alkyl alkyl ether sulfuric acid, polyoxyethylene alkyl phenyl ether sulfuric acid, polyoxyethylene dialkyl allyl ether sulfuric acid, polyoxyethylene alkenyl allyl ether sulfuric acid, and salts thereof. The method of claim 2, wherein the method is at least one compound. The method according to any one of claims 1 to 3, wherein the D-amino acid oxidase is a porcine kidney-derived D-amino acid oxidase. A D-amino acid oxidase-containing solution containing dithiodinicotinic acid. Furthermore, the D-amino acid oxidase containing liquid of Claim 5 containing an anionic surfactant. A reagent for measuring D-amino acid comprising the D-amino acid oxidase-containing solution according to claim 5 or 6. A kit for measuring homocysteine, comprising a reagent containing a thiol compound, homocysteine methyltransferase, and D-methionine methylsulfonium, and the reagent for measuring D-amino acid according to claim 7.

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