JP2000228998A - Homocysteine measurement using enzyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply and easily measure homocysteine useful for the diagnosis of arteriosclerosis, or the like by detecting a reaction product formed under action of both a nucleophilic reagent capable of substitution of a mercapto group located at the γ-position of homocysteine and an enzyme catalyzing the substitution reaction. SOLUTION: Homocysteine applicable to a versatile measuring instrument for clinical analysis and useful for the diagnosis of arteriosclerosis, or the like is simply, easily and specifically measured by making both a nucleophilic reagent capable of substitution of a mercapto group located at the γ-position of homocysteine and an enzyme catalyzing the substitution reaction act on a specimen including homocysteine, and by detecting one of reaction products consisting of γ-substitution-α-aminobutyric acid and hydrogen sulfide. The nucleophilic reagent is e.g. an inorganic salt anion, an organic acid or its salt anion, a thiol group-containing compound or its thiolate anion, a hydroxyl group-containing compound or its alcoholate anion, or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a simple and specific method for measuring homocysteine which is applicable to a general-purpose measuring instrument for clinical examination and is useful for diagnosing arteriosclerosis, and a kit for measuring the same.

[0002]

2. Description of the Related Art Homocysteine is an amino acid having a molecular weight of 135 existing in a living body, and is a risk factor already known as a risk factor for vascular disorders associated with arteriosclerosis, such as smoking, blood pressure, blood sugar, cholesterol and the like. Are emerging as uncorrelated, independent factors associated with atherosclerosis (New England Journal)
of Medicine, 324, 1149-11
55 (1991)). It is also known as an indicator of folate and cobalamin (vitamin B12) deficiency (New
England Journal of Medic
ine, 318, 1720-1728 (198
8), JP-A-7-49348).

[0003] Most of the homocysteine in blood is
It forms an SS bond with a mercapto group (SH group) present in the plasma protein, and is present in combination with the plasma protein, and its ratio is about 80 to 90% of the total homocysteine. In addition, endogenous low-molecular-weight compounds having SH groups, for example, those which are present as SS bonds with cysteine account for about 5 to 10% of the total homocysteine, and those in which homocysteine is present as SS bonds about 5%. -10%, and free homocysteine exists as about 1% of the total homocysteine.
And very few. Therefore, in the measurement of homocysteine, it is common practice to treat the specimen in advance with a reducing agent, and to release the SS-bound one as homocysteine before measuring.

[0004] Conventional methods for measuring homocysteine include gas chromatography mass spectrometry (Japanese Patent Application Laid-Open No.
221249, JP-A-7-49348) and liquid chromatography (HPLC) (Clinic
al Chemistry, 39, 1590-159.
7 (1993)), a method for separating and analyzing homocysteine, a fluorescent substance (Cli) via the SH group of homocysteine.
medical Chemistry, 35, 1921
-1927 (1989); ClinicalChemi
story, 39, 263-271 (1993)) and radioactive materials (Clinical Chemistry,
31, 624-628 (1985)), followed by separation and analysis of these derivatives by liquid chromatography. However, these methods require complicated pretreatment of the sample, require a dedicated analyzer, have a low sample processing capability, and have not been very accepted as clinical measurement methods.

The homocysteine is modified by a chemical method,
A method for immunoassay using an antibody specific to this homocysteine derivative (Japanese Patent Application Laid-Open No. 9-512634 (W
O95 / 30151)), homocysteine and adenosine are converted to S-adenosylhomocysteine hydrolase (EC
After the treatment in 3.3.1.1), the amount of adenosine changed in this reaction is detected by combining an enzyme reaction system or immunologically using an anti-adenosine antibody to indirectly detect the amount of homocysteine. Method for measuring the concentration (Tokuhei Hei 8-5064)
No. 78 (WO93 / 15220) is also known. However, these measurement methods also have some problems, such as complicated operations and a special immunoassay device.

Further, homocysteine is decomposed by using an enzyme, homocysteine desulfhydrase (EC 4.4.1.2), and hydrogen sulfide, ammonia, or 2-oxo of a product generated by this reaction is decomposed. A method of colorimetrically detecting any of butyric acid and measuring homocysteine is also known (WO98 / 07772). However, this enzyme has a problem with the specificity of the enzyme, such as reacting with methionine in addition to homocysteine, and is still incomplete. Furthermore, noting that the major form of homocysteine exists as a form that binds to plasma proteins, only human serum albumin-bound homocysteine was immunologically measured without treating the sample with a reducing agent. Attempts have been made to use a new diagnostic index (Japanese Patent Laid-Open No. Hei 10-11479).
No. 7) has not yet been established as a diagnostic index.

[0007]

DISCLOSURE OF THE INVENTION The present invention provides a simple and specific assay method using enzymes to enable homocysteine useful for diagnosing arteriosclerosis to be easily measured in clinical settings. An object of the present invention is to develop a kit for the measurement and apply the kit to a general-purpose measuring instrument for a clinical test which is widely used.

[0008]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, there is a reaction in which a nucleophile substitutes for an SH group in a homocysteine molecule. However, they have found that the use of an enzyme that catalyzes this enables simple and specific measurement of homocysteine, thereby completing the present invention.

That is, the present invention provides: (1) a sample containing homocysteine is reacted with a nucleophile capable of substituting for the γ-mercapto group of homocysteine and an enzyme catalyzing the substitution, A method for measuring homocysteine in a sample, which comprises detecting any of γ-substituted-α-aminobutyric acid and hydrogen sulfide to quantify homocysteine; (2) an enzyme catalyzing the substitution; The method according to the above (1), which is a γ-substituted-α-aminobutyric acid synthetase; (3) the method according to the above (1) or (2), wherein hydrogen sulfide is detected to determine homocysteine; 4) The nucleophilic reagent capable of substituting for the γ-mercapto group of homocysteine is an anion of an inorganic salt, an organic acid compound, an anion of a salt of an organic acid compound, a compound having a thiol group, or a thiolate of a compound having a thiol group. (5) γ of homocysteine, which is a nucleophilic reagent selected from the group consisting of an anion, a compound having a hydroxyl group, and an anion of an alcoholate of a compound having a hydroxyl group; A kit for measuring homocysteine comprising a nucleophile capable of substituting for a mercapto group, and an enzyme catalyzing the substitution; and (6) further comprising a releasing agent for releasing a homocysteine conjugate to homocysteine, The above (5)
The kit for measuring homocysteine described above.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
Samples of interest in the present invention include serum, plasma, cerebrospinal fluid,
In addition to saliva, urine, clinical samples such as extracts of erythrocytes, stool, tissues, cultured cells, etc., and industrial products such as foods, quasi-drugs, and pharmaceuticals, and these products when these products are solid In the case of an industry in which a product is manufactured by using a product extract or fermentation, a fermentation solution required for process inspection may be used.
In addition, homocysteine in a sample is easily condensed by an oxidation reaction between SH groups or an exchange reaction with an SS bond. Therefore, when homocysteine is present as a conjugate by binding to a low-molecular compound or protein having an SH group. There are many. For this reason, when it is necessary to measure homocysteine including these conjugates present in the sample, homocysteine is forcibly released from the conjugate by a chemical reaction or an enzymatic reaction, and this treatment is performed. The homocysteine in the solution is measured as a sample. For example, in a clinical specimen,
This is the case when "total homocysteine in plasma" is measured.

As homocysteine releasing agents for measuring homocysteine in a sample including conjugates, those reported by Jocelyn (Me
things of Enzymology, 143, 2
43-256 (1987)). Such releasing agents include, for example, dithiothreitol (DTT),
Low molecular weight compounds having an SH group, such as dithioerythritol (DTE), 2-mercaptoethanol, reduced glutathione, thioglycolic acid, cysteine, and various thioalcohols, may be mentioned. It is preferable to add an excessive amount of these releasing agents and gently release homocysteine by utilizing the SS exchange reaction with the releasing agents. For example, when measuring the total homocysteine in the plasma, for example, a release agent containing 1 to 20 mM DTT may be added to the plasma and reacted at room temperature for 30 minutes.

The nucleophile capable of substituting for the γ-mercapto group of homocysteine used in the present invention is not particularly limited as long as it is a suitable substrate for an enzyme which catalyzes the substitution reaction with the γ-position SH group in the homocysteine molecule. May be used, and there is no particular limitation. For example, an anion of an inorganic salt, an organic acid compound, an anion of a salt of an organic acid compound, a compound having a thiol group, a thiolate anion of a compound having a thiol group, a compound having a hydroxyl group, an anion of an alcoholate of a compound having a hydroxyl group, and the like. can give.

The anions of the inorganic salts include, for example, cyanide ion, azide ion, isocyanate ion, thiocyanate ion, bromide ion, iodide ion, sulfide ion, sulfite ion, phosphate ion and the like. Examples of the anion of the organic acid compound and salts of the organic acid compound include organic acid compounds such as acetic acid and trifluoroacetic acid, and anions of salts of these organic acid compounds. Examples of the compound having a thiol group and the anion of the thiolate of the compound having a thiol group include DTT, DTE, 2-mercaptoethanol, reduced glutathione, thioglycolic acid, cysteine, N-acetylcysteine and thiophenol. Or thioalcohol derivatives of aromatic compounds, and anions of thiolates of these thioalcohol derivatives. Examples of the anion of the compound having a hydroxyl group and the alcoholate of the compound having a hydroxyl group include alcohols such as methanol and ethanol; ethylene glycol, polyols such as glycerin and glucose; aromatic hydroxy compounds such as phenol; and these hydroxyl groups. And an anion of an alcoholate of a compound having the same. Among the nucleophiles capable of substituting the γ-position mercapto group of homocysteine exemplified above, DT
T, DTE, 2-mercaptoethanol, reduced glutathione, thioglycolic acid, cysteine and the like can also be used as a releasing agent for releasing homocysteine from a homocysteine conjugate. The counter ion of these nucleophilic reagents may be any as long as it does not hinder homocysteine measurement, and is not particularly limited. In addition, components that are difficult to dissociate as anions do not need to be added as ions and can be used as they are, provided that they act as nucleophiles in the reaction.

The enzyme used in the present invention that catalyzes the reaction of substituting the mercapto group at the γ-position of homocysteine with a nucleophile includes homocysteine desulfhydrase (EC 4.
Unlike an enzyme that degrades homocysteine such as 4.1.2), any enzyme capable of catalyzing the substitution reaction of the following reaction formula I may be used, and is not particularly limited. . γ-SH-α-aminobutyric acid (homocysteine) + nucleophile (A ⁻ ) + H ⁺ → γ-A-α-aminobutyric acid + hydrogen sulfide (H ₂ S) Reaction formula I

Such enzymes include, for example, γ-substituted-
α-aminobutyric acid synthetase. γ-substituted-α-
Aminobutyric acid synthase is defined by the following reaction formula II (reaction formula II).
X in the above represents a substituent such as an SH group, and YH represents an acid type of the anionic species Y. ) Means an enzyme that catalyzes the reaction shown in (a) to produce the compound “γ-substituted-α-aminobutyric acid” of (a). γ-X-α-aminobutyric acid + YH → γ-Y-α-aminobutyric acid (a) + XH Reaction formula II This γ-substituted-α-aminobutyric acid synthase is an enzyme widely existing in the microorganism world. And EC. 4.2.99.9 or E
C. 4.2.99.10, and γ-cyano-α-aminobutyric acid synthase that catalyzes the reaction of the following reaction formula III, and the like. O-acetyl-L-homocysteine + HCN → γ-cyano-α-aminobutyric acid + acetic acid Reaction formula III

These enzymes can be easily obtained from the market, or they can be obtained by directly purifying them from microorganisms or animals that produce the enzymes. In addition, the genes encoding these enzymes are taken out, and the genes or genes modified with reference to the gene information and genes created with reference to the amino acid sequence information of the enzymes are expressed using genetic engineering techniques. Thus, enzymes can be produced. In addition, those enzymes obtained by modifying these enzymes while maintaining the enzyme activity can be similarly used, and such enzymes are also included in the enzymes used in the present invention. Examples of microorganisms having γ-substituted-α-aminobutyric acid synthase activity include, for example, Baci
llus coagulans IAM1115, Bacillus stearothermophilu
s ATCC21365, Salmonella typhimurium IFO12529, Micr
ococcusluteus IFO3066, Brevibacterium ammoniagenes
IFO12071, Pseudomonas putidaIFO12653, Nocardia er
ythropolis IFO12539, Rhodococcus erythropolis IFO1
2538, Rhodococcus erythropolis IFO12540 and the like.

When the total homocysteine in a sample is measured using an enzyme that catalyzes the reaction of substituting the γ-mercapto group of homocysteine with a nucleophile according to the present invention, for example,
1-10000 units / ml, preferably 1-1000 mM in the presence of 0.1-1000 mM, preferably 1-100 mM nucleophile.
PH 3.5-11.5, using 300 units / ml enzyme
The reaction may be carried out preferably in a buffer solution of pH 5-10 at 5-50 ° C, preferably 25-45 ° C for 1-10 minutes.
The buffer is selected from those having a buffering action in the above-mentioned pH range, and is a buffer derived from an inorganic acid salt such as Good buffer, Tris buffer, phosphoric acid, carbonic acid or boric acid, or an organic acid such as acetic acid or citric acid. Buffers derived from salts and the like can be mentioned. When a sample such as serum or plasma containing a homocysteine conjugate is used, homocysteine may be previously released using a releasing agent, but the above-mentioned DTT, DTE, Using a nucleophile such as mercaptoethanol, reduced glutathione, thioglycolic acid, or cysteine, the release of homocysteine from the conjugate in the sample and the substitution reaction with the nucleophile may be performed continuously.

The product of the enzymatic reaction of the present invention is defined by the reaction formula I
Γ-A-α-aminobutyric acid (A is a substituent by the nucleophile A anion) and hydrogen sulfide. As a method for detecting these products, HPLC
Induction of a substance having color, fluorescence, luminescence, etc. from these products by measurement using an enzyme reaction, chemical reaction, or a common method combining these reactions These substances can be detected with a luminometer. When these substances are oxidizing / reducing substances, they can be directly detected by an electrochemical detector.

In the present invention, the method for detecting hydrogen sulfide is not particularly limited as long as it is capable of measuring homocysteine present in a sample with specificity and sufficient sensitivity. When clinically measuring homocysteine in serum or plasma, a colorimetric method that can be applied to a general-purpose measuring instrument for clinical tests that is currently widely used is more preferable.

As a colorimetric method, for example, a method of reacting hydrogen sulfide with lead acetate to generate lead sulfide and measuring the absorbance (for example, 360 nm) of the generated lead sulfide (IRCS M)
electronic Science, 13, 493-49
4 (1985)), the oxidative condensation of hydrogen sulfide and N, N-dimethyl-p-phenylenediamine hydrochloride in the presence of zinc acetate and ferric chloride, and the absorbance of the resulting methylene blue (650-670 nm, molecular extinction coefficient) 26,000) (Oceanogr., 14, 454-).
458 (1969)), and a method of chemically producing a dye. In addition, sulfite ion reductase (E
C 1.8.1.2) and oxidized nicotinamide adenine dinucleotide phosphate (NA
DP) to give reduced form of NADP (NADPH), and furthermore, diaphorase and 1-methoxy-5-methylphenadium methyl sulfate (1-m-PM
In the presence of an electron carrier such as S), the tetrazolium salt is reduced with NADPH to form a formazan dye (570 nm, molecular extinction coefficient 15,000), and is measured by a combination of enzymes such as those measured from the absorbance of the dye. There is also a way to do it. For example, when measuring total homocysteine in plasma, its abundance is usually 1 to 200 μM, so that it is within a range that can be sufficiently measured even by these colorimetric methods.

The product of the enzymatic reaction in the present invention,
As a method for detecting γ-substituted-α-aminobutyric acid, H
Examples include a measurement by PLC, an enzymatic reaction, a chemical reaction, or a general method combining these reactions.

The kit for measuring homocysteine of the present invention comprises:
Basically, a nucleophile capable of substituting for the γ-mercapto group of homocysteine, and an enzyme catalyzing the substitution, for example, γ
-Substituted-α-aminobutyric acid synthase. However, the composition of the reagents in the kit may vary depending on the target analyte. That is, when it is necessary to measure also including a low molecular compound having an SH group or homocysteine conjugated to a protein, generally,
Further, a releasing agent for releasing the conjugated homocysteine can be added. In addition, a coloring agent for detecting hydrogen sulfide, which is a product of an enzymatic reaction, can be added. In constructing the kit for measuring homocysteine of the present invention using these reagents, when components that interfere with each other are present, the components can be divided and subdivided for each component. Conversely, if there is no problem in use even if these constituent reagents are integrated, they can be integrated to further simplify the constituent reagents. For example, the same compound can be used for the releasing agent and the nucleophile.

In the case of a kit for measuring total homocysteine using plasma as a sample, for example, a release agent containing DTT, γ-
It comprises a hydrogen sulfide generating agent containing cyano-α-aminobutyric acid synthase and a color former for detecting hydrogen sulfide as methylene blue. The amount of each component used per kit varies depending on the number of tests in the kit and the amount of reaction solution per test.
For example, when using DTT as the releasing agent, 0.02-2 mmol, for example, when using γ-cyano-α-aminobutyric acid synthase as the hydrogen sulfide generating agent, 20-4000. Unit. For example, when a cyano ion is used as the nucleophilic reagent for substituting the γ-mercapto group of homocysteine, 0.02-2 mmol of potassium cyanide is used. However, when a component having an SH group, for example, DTT is used as the releasing agent, it is not necessary to add a new nucleophile because DTT itself acts as the nucleophile.

[0024]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

Example 1 Determination of homocysteine for detecting hydrogen sulfide by the absorbance method
Homocysteine solution 100μL of each concentration as an amount analyte,
20 mM potassium cyanide and 100 U / mL of γ-cyano-α-aminobutyric acid synthase (Ikeda Saccharification Co., Ltd.)
10 mM potassium phosphate buffer (pH 7.5) containing 0.16 mM pyridoxal-5-phosphate (coenzyme of γ-cyano-α-aminobutyric acid synthase)
0 μL was added, and the mixture was reacted at 45 ° C. for 10 minutes. As a reagent for detecting hydrogen sulfide, 1.3 mL of a 1% aqueous solution of zinc acetate was added,
50% of 12% sodium hydroxide solution, 300μ of distilled water
L was added and the mixture was sealed and stirred. Then open the stopper and 1%
250 μL of a 5.5 M hydrochloric acid solution of N, N-dimethyl-p-phenylenediamine hydrochloride and 50 μL of a 1.2 M hydrochloric acid solution of 23 mM ferric chloride were quickly added and sealed again. After standing at room temperature for 30 minutes, 0.85 mL of distilled water was added, and the absorbance at 670 nm was measured. The relationship between the obtained absorbance and the concentration of homocysteine was plotted to prepare a calibration curve. As shown in FIG. 1, the calibration curve was a straight line from 1 μM to 400 μM, and the quantification of homocysteine was possible.

Example 2 γ-cyano-α-aminobutyric acid is detected by HPLC method
100 μL of each concentration of homocysteine as a homocysteine quantitative sample,
100 μL of 100 mM potassium phosphate buffer (pH 7.5) containing 20 mM potassium cyanide, 0.16 mM pyridoxal-5-phosphate and 100 U / mL γ-cyano-α-aminobutyric acid synthase were added, and 45 mL of the mixture was added.
After reaction at 10 ° C. for 10 minutes, the reaction was stopped by placing it in a boiling water bath for 2 minutes. Subsequently, the precipitate was removed by centrifugation at 15,000 rpm for 5 minutes, and the supernatant was subjected to HPLC to quantitate γ-cyano-α-aminobutyric acid generated by the enzyme reaction. The HPLC conditions were as follows:
-2 packed column (inner diameter 4.6 x length 250mm)
The mobile phase was carried out using an eluent of 20 mM sodium phosphate buffer (pH 6.8) and acetonitrile in a ratio of 85:15. The relationship between the homocysteine concentration and the chromatographic peak area value of γ-cyano-α-aminobutyric acid in HPLC was plotted to prepare a calibration curve. FIG.
As shown in the figure, the calibration curve was linear from 1 μM to 400 μM, and homocysteine was quantified.

Example 3 Specificity of the homocysteine assay of the present invention In order to confirm the specificity of the assay system of the present invention for homocysteine, an aqueous solution of homocysteine, methionine, glutathione and dithiothreitol at a concentration of 10 mM was used as a sample. The measurement was performed by the methods of Example 1 and Example 2, and the measured values as homocysteine were compared. As shown in Table 1, this assay did not react with methionine and dithiothreitol at all, and was specific to homocysteine.

[0028]

Table 1 Specificity of homocysteine measurement method of the present invention Measured value as homocysteine (mM) Substrate absorbance HPLC method homocysteine 10.0 (100%) 10.0 (100%) Methionine 0 (0 %) 0 (0%) Glutathione 0.5 (5%) Trace peak dithiothreitol 0 (0%) Not measured

Example 4 Measurement of Homocysteine in Plasma To 100 μL of plasma, 10 μL of a 0.1 M DTT solution was added, mixed, and allowed to stand at room temperature for 30 minutes. 0.08
100 μL of 50 mM potassium phosphate buffer (pH 7.5) containing mM pyridoxal-5-phosphate and 10 U / mL of γ-cyano-α-aminobutyric acid synthetase was added, and the mixture was reacted at 45 ° C. for 10 minutes. As a hydrogen sulfide detection reagent, 520 μL of a 1% aqueous zinc acetate solution, 20 μL of a 12% sodium hydroxide solution, 100 μL of a 0.1% N, N-dimethyl-p-phenylenediamine hydrochloride solution,
Add 20 μL of a 3 mM ferric chloride / hydrochloric acid solution, and add
After reacting for 1 minute, 340 μL of distilled water was added, and the absorbance at 670 nm was measured. Reagent blinding was performed in the same manner using distilled water instead of the DTT solution. The homocysteine concentration in plasma was determined using a homocysteine standard solution.
It was determined using a calibration curve created by measurement. As a control, the SH group of homocysteine was fluorescently labeled, and
Analyzed by JB. Ublink et al.'S method (J. Chr.
omatogr. , 565, 441-446 (199).
The same plasma was measured according to 1)). These results are summarized in Table 2 and compared. As shown in Table 2, the values measured by the enzymatic method of the present invention were in good agreement with the values of the HPLC method and proved to be reliable.

[0030]

Table 2 Comparison of plasma homocysteine measurement homocysteine concentration (μM) Sample No. Enzymatic method of the present invention Known HPLC method 1 15.6 14.8 2 11.5 10.6 3 8.7 7.9 4 10.1 10.5 5 14.3 13.2 6 26.2 25.9 7 18.3 17.8 8 12.6 12.2 9 11.5 10.2 10 10.6 8.6

[0031]

According to the measuring method of the present invention, the homocysteine concentration can be measured only by adding a reagent to a sample. Using this method, homocysteine in a clinical sample can be easily measured with a general-purpose device for clinical tests. Therefore, the measurement method of the present invention is useful for diagnosing arteriosclerosis and can contribute to treatment and control of adult diseases.

[Brief description of the drawings]

FIG. 1 shows a calibration curve of a homocysteine measuring method for detecting hydrogen sulfide according to the present invention.

FIG. 2 shows a calibration curve of a homocysteine measuring method for detecting γ-cyano-α-aminobutyric acid according to the present invention.

 ────────────────────────────────────────────────── ─── Continued on the front page F term (reference) 2G045 AA13 AA25 AA28 CA25 CA26 DA35 FB01 FB05 FB11 GC12 4B063 QA01 QA19 QQ03 QQ15 QQ16 QQ17 QQ61 QQ80 QQ89 QR18 QR41 QR65 QS17 QS28 QX01

Claims (6)

[Claims] 1. A nucleophile capable of substituting a homocysteine-containing specimen with a γ-mercapto group of homocysteine,
And reacting an enzyme that catalyzes the substitution, wherein any of γ-substituted-α-aminobutyric acid and hydrogen sulfide, which are products of the enzymatic reaction, is detected to quantify homocysteine in the sample. Method for measuring homocysteine. 2. The enzyme which catalyzes the substitution is γ-substituted-α-
The method according to claim 1, which is an aminobutyric acid synthase. 3. The method according to claim 1, wherein homocysteine is quantified by detecting hydrogen sulfide. 4. A nucleophilic reagent capable of substituting for a γ-mercapto group of homocysteine is an anion of an inorganic salt, an organic acid compound, an anion of a salt of an organic acid compound, a compound having a thiol group, or a compound having a thiol group. The method according to any one of claims 1 to 3, which is a nucleophile selected from the group consisting of a thiolate anion, a compound having a hydroxyl group, and an anion of an alcoholate of a compound having a hydroxyl group. 5. A kit for measuring homocysteine, comprising a nucleophile capable of substituting for a γ-mercapto group of homocysteine, and an enzyme catalyzing the substitution. 6. The kit for measuring homocysteine according to claim 5, further comprising a releasing agent for releasing a homocysteine conjugate into homocysteine.