JPS5836400A - Determining method of l-cysteine - Google Patents

Abstract

PURPOSE:A method of determining the titled substance, by reacting glutathione sulfhydryl oxidase with a sample in the presence of oxygen, and measuring the oxygen consumption or the amount of the formed reaction product, e.g. hydrogen peroxide. CONSTITUTION:Glutathione sulfhydryl oxidase (GSO) is reacted with a sample containing substantially no glutahione in the presence of oxygen, and the amount of the formed reaction product, e.g. hydrogen peroxide, or oxygen consumption is measured to determine L-cysteine. In the case of a sample containing glutathione, the enzymic reaction of GSO is carried out at 7-8pH and 4.5-5.5pH, and the content of the L-cysteine is obtained from the difference in the oxygen consumption or the amount of the formed reaction product the reaction solution between both pH values. This is because the GSO is reacted with the glutathione at 4.5-5.5pH to oxidize the gluhathione and further L-cysteine at 7-8pH.

Description

DETAILED DESCRIPTION OF THE INVENTION [1] Background of the Invention The present invention is directed to converting L-cysteine (hereinafter abbreviated as L-Cys) into a new enzyme, glutathione sulfhydryl oxidase (hereinafter abbreviated as GSO). It relates to a method for quantitative determination using

Conventionally, methods for quantifying L-(:ys include the acidic ninhydrin method or chemical methods using reagents that react with SH (thiol) groups such as 5,5'-dithiobis-(2-nitrobenzoic acid) and 0-phthalaldehyde. Although methods are known, no method has been established that is necessarily satisfactory in terms of specificity, simplicity, sensitivity, or influence of contaminants.

The present inventors previously discovered that a new sulfhydryl oxidase, GSO, which is highly specific for glutathione (hereinafter abbreviated as GSH), is produced by microorganisms, and investigated the enzymatic properties of this enzyme. revealed (
(See Japanese Patent Application No. 16998/1982).

As a result of further research to apply this enzyme to the quantification of L,-Cys, the present inventor discovered that L-Cys in the sample can be quantified by GS even if other SH compounds such as GsH coexist.
The present inventors have discovered that by utilizing the change in specificity of O due to I)H, it is possible to easily quantify specific ζ with high sensitivity, and have completed the present invention.

(II) Summary of the invention! j The present invention is characterized in that when quantifying L-Cys in a sample, GSO is applied to the sample in the presence of oxygen, and the amount of oxygen consumed or the amount of reactant produced accompanying the reaction is measured. It provides a quantitative method. Especially G
When quantifying L-Cys in a sample containing SH, G
The enzymatic reaction with SO was performed using I) H7,0-8.0 and p
Oxygen consumption (Al or reactant production amount (A
Then, measure the oxygen consumption fBl or the amount of reactant produced (decoy) in the reaction solution at pH 4.5 to 5.5, and calculate the L -Cys content from the difference (A-B or △'-■). The present invention provides a method.

t The GSO used in the present invention has high specificity for GSH at pH 4.5 to 55, and if the optimal enzyme amount is used, it almost specifically oxidizes only GSH among the S'H compounds present in the sample. However, 1) In H7,0-80, if a sufficient amount of enzyme is present, L together with GSH
-Cys can also be completely oxidized. Therefore, in the case of a sample such as a biological sample where most of the existing SH compounds are occupied by GSH and L-Cys, the oxygen consumption (Al
Or the amount of reactant produced (Xl to I), the amount of oxygen consumed in H4,5-5.5 (B) or the amount of reactant produced (by subtracting the decoy, only the amount of L-Cys in the sample can be easily and accurately quantified) In addition, L in the sample can be
It goes without saying that when SH compounds other than -Cys, particularly GSH, are substantially absent, L -Cys can be quantified by simply measuring at pH 7.0 to 80.

The enzymatic chemical and physicochemical properties of the purified enzyme preparation of GSO in the present invention are as follows.

(1) Action GSO is a known sulfhydryl oxidase from rat seminal vesicle secretion (Biochem
istry)19. 2689-2645 (1980)
(Reference), two molecules of SH compound are oxidatively condensed in the presence of an enzyme to produce the corresponding disulfide compound, but it shows particularly strong affinity for GSH and high substrate specificity, and also has a high affinity for SH groups in proteins. It is a novel sulfhydryl oxidase that exhibits only weak activity against.

When GSH is used as a substrate, GSO requires 1 mol of oxygen per 2 mol of G S H, and 1 mol of oxidized glutathione and 1 m
ol and hydrogen peroxide.

Furthermore, when L-CyS is used as a substrate, as in the case of GSH, 1 mol of oxygen is required per 2 mol of l, -Cys, and 1 mol of cystine and 1 mol of hydrogen peroxide are produced.

2R5H+02→R55R+H2O2 (wherein, R5H is GSH or L-Cys, R35
R represents oxidized glutathione or cystine. )(2
) Substrate specificity Purified GSO for various SH compounds was acidified (p
H5,0; 0.2M acetate buffer), neutral (pH7,4;
0.2M phosphate buffer) and alkaline (pH 9,8
Table 1 shows the results of the reaction at various pH values of 0.2M) Squirrel buffer. In Table 1, the concentration of each substrate is 5
It is mM. Further, the enzyme activity was measured by the oxygen electrode method described later, and expressed as a relative value of the activity against GSH at each pH.

As is clear from Table 1, GSO showed high substrate specificity for GSH at pH 5, (i). At pH 7.4, it efficiently acted on L -Cys and dithiothreitol in addition to GSH. However, when acting on biological samples, there is virtually no dithiothreitol, so
If a sufficient amount of enzyme is used, L-Cys as well as GSH can be completely oxidized. Km at pH 7.4
As a result of measuring the value, the Km value for 'G S H was 6.
．． The Km value for L-Cys was 8.7'X-10-8M.

Note that GSO is clearly distinguished from the known sulfhydryl oxidase from rat seminal vesicle secretion, which exhibits high substrate specificity and strong affinity for dithiothreitol.

In addition, GSO has almost no effect on the SI group of proteins, and could be distinguished from known enzymes in this respect.For example, GSO was reduced in 2-mercaptoethanol in the presence of 8M urea, 1870
When GSO was allowed to act on the SH groups of 0), even when a sufficient amount (5 Uy'vil) of GSO was added, the rate of decrease in SH groups (8 pieces) per molecule of RNa8e A was within 4 hours. The number of s due to natural oxidation is about 1.5.
This rate was slightly higher than the rate of decrease in H groups (1.0 units/4 hours). On the other hand, when 0.5 U/gl of known sulfhydryl oxidase in rat seminal vesicle secretion is added, it shows a decrease of more than 7.5 cells/mol in 4 hours.
(1980), Fig. 7), is an enzyme that exhibits significant activity against RNASeA.

That is, the GSO of the present invention has GSH on the acidic and neutral sides.
It is a novel flavoprotein sulfhydryl oxidase that exhibits strong affinity for and specifically acts on flavoprotein sulfhydryl oxidase.

(8) Measurement of titer The titer of GSO was measured using an oxygen electrode method.

That is, 0.0000 mg containing 10 mM GSH. 1 g of IM potassium phosphate buffer (pH 7,4) was placed in an oxygen electrode cell, 107z5 enzyme solution was added, and the oxygen consumption rate was measured. The amount of enzyme that consumed 1 μmo+ oxygen per minute at 80° C. was defined as 1 unit (Unit H, herein abbreviated as U).

(4) Optimal pH Optimum 1) H is around pH 6.5 to 77, especially pH 7.1 to
It is around 7.8.

The optimum pH is measured using 0.15M sodium acetate.
Hydrochloric acid buffer, 0.15M sodium acetate-acetate buffer, 0.15M potassium phosphate buffer, 0.15M) Lis-HCl buffer, and 0.15M glycine-sodium chloride-sodium hydroxide buffer. pH
It was carried out in the range of 2.4 to 10.8.

f51 pH stability and thermal stability The pH stability was determined at pH 8.0 to 10 in each of the above buffer solutions.
．． 8, the enzyme activity was measured after holding at 45°C for 115 minutes, and it was found to be stable in the pH range of 5 to 9.

In addition, thermal stability was determined using 0.1M potassium phosphate buffer (p
The enzyme activity was measured by holding at 40 to 80°C for 15 minutes in H7,4). The results showed that the activity was completely maintained up to 55°C, but completely inactivated at 800°C.

(6) Range of suitable temperature for action In the oxygen electrode method used to measure the enzyme activity of GSO, the amount of dissolved oxygen in the reaction solution varies depending on the temperature, so it is difficult to determine the exact range, or the generation of hydrogen peroxide is As measured by the method, a range of approximately 30 to 50°C was suitable.

(7) Inhibition, activation and stabilization potassium cyanide, calcium chloride, potassium chloride, nickel chloride, aluminum chloride, strontium chloride,
Various metal salts such as barium chloride, ferric chloride, magnesium sulfate, zinc sulfate, manganese sulfate, copper sulfate, lithium sulfate, potassium iodide, and ethylenediaminetetraacetic acid (
Enzyme reactions were performed in 0.1 M potassium phosphate buffer (+)H7,4) containing 1 mM of an inhibitor such as EDTA), and the presence or absence of inhibition by each substance was examined. As a result, G.S.
The activity of O was completely inhibited by zinc sulfate, that is, zinc ions, and about 20% by manganese sulfate, but hardly inhibited by other inhibitors.

(8) Ultraviolet absorption spectrum λmax 272nm, 365nm,
443nmE280nm14.78 1% (9) The supernatant obtained by heat treatment or trichloroacetic acid (TCA) treatment of coenzyme GSO and centrifugation has an absorption spectrum that matches that of flavin adenine dinucleotide (FAD), and D-
Since the apoenzyme of amino acid oxidase was activated, the coenzyme of this enzyme was found to be FAI). Also,
FA'D exists in 2m01 per G501m01.

+IQ polyacrylamide gel electrophoresis and 5D
S-polyacrylamide gel electrophoresis Purified enzyme showed a single band by both methods.

The pI was measured by isoelectric focusing using ampholine (manufactured by LKB, Sweden), and the pI was 4.21.

(The molecular weight of 19 molecular weight GSO was determined to be 94000±5000 by gel filtration using Sephadex G-200 (manufactured by Pharmacia Fine Chemicals).
Polyacrylamide gel electrophoresis showed that GSO is a dimeric enzyme composed of two subunits with a molecular weight of 47,000±3,000.

(13) Crystal structure and elemental analysis GSO was not measured because it was not crystallized.゛(4) Purification method' GSO can be purified by salting out method, isoelectric precipitation method, precipitation method using organic solvent, adsorption method using diatomaceous earth, activated carbon, etc.
Various chromatographic methods can be used in combination as appropriate. Specific examples of the purification method are as shown in Reference Examples.

2) Production of GSO The GSO used in the present invention is not limited by its origin or production method, as long as it has the above-mentioned properties. As an example of a manufacturing method, G by microorganisms
A method for producing SO will be specifically shown as a reference example.

Reference example 89 bran and 5 m water in an 800vtl Erlenmeyer flask
Penicillium acreatum IFO5729 was inoculated into a bran culture medium prepared by adding 1 g of rice husks and 1 g of rice husk and autoclaving at 120°C for 80 minutes, and culturing at 28°C for 7 days to prepare a seed culture.

Twenty 51-volume Erlenmeyer flasks were each filled with 200 liters of wheat bran and 140 liters of water, sterilized under pressure at 120°C for 30 minutes, and then aseptically inoculated with the above-mentioned inoculum and cultured at 28°C for 7 days. The obtained culture was immersed in 8011 water for 1 hour, filtered, and further passed through diatomaceous earth to obtain crude enzyme 274.

Ammonium sulfate was added to this crude enzyme solution up to 65%, and the produced insoluble matter was removed by centrifugation. Ammonium sulfate was further added to the supernatant to achieve 95% saturation concentration, and the resulting precipitate was collected by centrifugation and added to 0.02 M phosphate buffer (+) H7.
, 4) 11 and dialyzed overnight against the same buffer. The precipitate generated during dialysis was removed by centrifugation, and the supernatant was passed through a DEAE (diethylaminoethyl)-cellulose column (5X70α) equilibrated with the same buffer, and the adsorbed enzyme was purified with the same buffer containing 0.2 M of sodium chloride. It was eluted using The eluted active fraction was collected, and after dialysis and concentration, Sephadex G~1
00 (manufactured by Pharmacia Fine Chemicals) column (8,5 x 100z) was used for repeated filtration, and the active fraction was collected and concentrated. Dialyzed. This dialysis fluid is centrifuged,
After microfiltration of the supernatant synovial fluid, it was freeze-dried to obtain a purified sample of GSO (yield 41%, specific activity 540 U/my protein) 49~
I got it.

Microorganism used The microorganism used in the production of GSO of the present invention is Aspergillus/l/x (hereinafter referred to as A
It is abbreviated as. ] Genus, Penicilli
um ;hereinafter referred to as P.

It is abbreviated as. ), genus Fusarium
; Hereinafter, it will be abbreviated as F. ) genus, Tory y Chill 7 (
Trichoderma; hereinafter abbreviated as T. ), genus paecilomyces; p
It is abbreviated as ae. ) or Gliocladium (Ql
iocladium; hereinafter abbreviated as G. ) and is a microorganism that has GSO-producing ability. Specific examples of GSO-producing bacteria belonging to the genus Stomach include Aspergillus awamori (A, awamori) IAM 22
99. Aspergillus batatae (A, batata)
e) IAM 2098, Aspergillus inui (A, inui) IAM 2255, Aspergillus ochraceus x (A, ochraceus
) IF 04345, Ashergi JLt7. - Penicillium aculeatum (A, oryzae) IFO4188, Penicillium aculeatum (p, aculeatum)
I F 0 5? 29, Penicillium jenseni (P, jenseni) I AM 7197,
Henicillium citrinum tom 1181 (Pcit
rinum Thom 1181)
No. 868, Fusarium solani (F, 5olani)
) I F 05282, Fusarium sambusinum (F.

sambucinum) OU T 4020, Gliocladium teriquescens (G, dellque
CenS) OUT4616, Baecilomyces paevarioti I F O4855, Trichoderma viride (T, viride) ATCC205
86tx etc. However, the microorganisms used in the present invention are not limited to these strains. In addition, these GSO-producing bacteria were mutated by conventional microbial mutation induction methods, such as physical treatments such as ultraviolet rays, X-rays, and R-ray irradiation, and chemical treatments with drugs such as nitrosoguanidine. Any GSO high-producing mutant strain can be suitably used.

Preparation of GSO The preparation form and degree of purification of GSO are appropriately selected depending on the oxidation reaction by the present enzyme and the type of subsequent detection system for the indicator substance. In other words, GS
Even if there are contaminants in the O enzyme preparation, problem 1 cannot be solved. Furthermore, depending on the embodiment of the quantitative system employed, the enzyme can be used as a soluble enzyme, or as an enzyme adsorbed or insolubilized on a suitable carrier.

3) Quantification of L-cys The method for quantifying L-Cys in the present invention is to quantify L-cys in a sample.
-(::ys oxidation reaction by GSO, especially when targeting GSH coexisting samples, pH 4.5 to 5.5 and p
It consists of a specific oxidation reaction at two points, H7.0 to H8.0, and a detection system for an indicator substance that measures the amount of oxygen consumed or the amount of reactant produced.

Enzyme reaction using GSO The conditions for the GSO enzyme reaction in the determination of L-CY8 are as follows. That is, the reaction pH is GSO stable p
The pH range may be within the H range and act on L -Cys, but especially when L -Cys in the GSH coexisting sample is
When measuring pH 4.5 to 5.5 and pH 7.0
1) Must be in the H range of ~8.0. The change in substrate specificity due to pH of GSO can only be utilized by carrying out the reaction in both pH ranges. In the above case,
The GSO of the Vanicillium filamentous fungus shown in the reference example is particularly p.
By conducting the reaction at around H5.0 and pH around 7.4, changes in substrate specificity due to pH can be suitably utilized.

In addition, when targeting a sample in which GSH coexists, the amount of enzyme used relative to the substrate is such an excess amount that L-Cys and GSH completely react in a reaction at pH 7.0 to 8.0. In the reaction at pH 4.5 to 5.5, it is preferable to use an amount sufficient to oxidize GSH. Further, in order to maintain the pH of the enzyme reaction, it is preferable to use various buffer solutions as the enzyme reaction medium. The buffer solution should be one that can maintain the above pH range and does not inhibit the enzyme reaction, such as ilF! - Buffers are preferred.

The reaction temperature is not particularly limited as long as it is within the stable temperature range of GSO.

Detection of Indicator Substance The indicator substance in the present invention is oxygen and a reactant (reaction product) in an enzyme reaction. The reactants in the GSO reaction are hydrogen peroxide and a disulfide dimer corresponding to the substrate S 2 H compound, and hydrogen peroxide is suitable as the indicator substance of the present invention.

Quantification of L, -Cys is carried out by measuring the change in the amount of the indicator substance accompanying the enzymatic reaction.

Specifically, as a method that can be applied regardless of the presence or absence of GSH in the sample, the amount of oxygen consumed (Al or the production of one of the reactants, such as hydrogen peroxide) accompanying the enzymatic reaction at pH 7.0 to 8.0 is amount fAj and pH 4.5 to 5.
The amount fBl of oxygen produced due to the enzymatic reaction in step 5 or the amount produced of one of the same reactants as described above (by detecting and measuring the angle,
A method for quantifying L-Cys is by determining the difference between constant values on both sides (A-B or A'-B').

Of course, when targeting a sample that clearly does not substantially contain GSH, the enzymatic reaction is performed at a pH at which L-cys reacts sufficiently, for example, I) H7.0 to 8.0;
L by measuring the amount of oxygen consumed or the amount of reactants produced
-C'ys can be easily quantified.

The detection system for the indicator substance can be selected arbitrarily, and methods commonly used for measuring these substances can be applied.

Furthermore, there is no particular limitation on the specific method for detecting the indicator substance.

Representative methods for detecting and measuring each indicator substance will be illustrated below, but the detection systems for these indicator substances are not limited to known methods, and will continue to be developed in the future. It is also considered possible to adopt

Warburg's pressure detection method and oxygen electrode method are known methods for measuring oxygen consumption (Biochemistry Experiment Course 5).
Enzyme research methods (1) pp. 35-52, Tokyo Kagaku Doujin,
(Published August 20, 1975).

Furthermore, an enzyme electrode method in which GSO is immobilized on the tip of an oxygen electrode can also be adopted.

There are three main methods for detecting and measuring hydrogen peroxide: spectroscopic measurement, fluorescence measurement, and electrochemical measurement.

Spectroscopic methods for measuring hydrogen peroxide include methods using a combination of a hydrogen peroxide activator and an indicator.

As the hydrogen peroxide activator, in addition to peroxidase such as horseradish or sweet potato, any one of iodide, molybdate, etc. or a mixture thereof can be effectively used. As an indicator, 0-
Dianisidine, 0-tolidine, 0-toluidine, 2.6
-dichloroindophenol, benzidine, 8.8'
In addition to -5,5'-tetraalkylbenzidine (such as 8,8'-dimethyl-5,5'-diethylbenzidine) and 4-methoxy-1-naphthol, combination indicators can be used. Examples of combination indicators include 4-
A combination of aminoantipyrine and phenol may be mentioned, but is not particularly limited to this. For example, instead of phenol, polyhydric alcohols or phenol derivatives such as 2,4-dichlorophenol, catechol, resorcinol, hydroquinone, cresol, guaiacol, pyrogallol, orcinol, and also aniline such as aniline, dimethylaniline, diethylaniline, etc. Derivatives can be used. On the other hand, examples of substitutes for 4-aminoantipyrine include 4-aminophenazine, various 4-aminopyrazolone derivatives, 3-methylbenzothiazolinone-hydrazone (MBTH) and its sulfonic acid derivatives. In addition to the above-mentioned indicators, any indicator can be used as long as it can be quantitatively oxidized under quantitative conditions and exhibit a change in color tone. Other spectroscopic methods for measuring hydrogen peroxide include forming formaldehyde from hydrogen peroxide in the presence of methanol and catalase, which is then combined in the presence of an oxidizing agent (e.g., potassium cyanophelate, ferric chloride, etc.). There is a method of reacting with hydrazone (for example, 3-methyl-2-(sulfonyl)-benzothiazolone hydrazone). Furthermore, the catalase-acetylacetone method, the method by oxidation of alcohol using aldehyde dehydrogenase,
A method is known in which indigo carmine is oxidized and decolorized using a copper ion-histamine system and quantitatively determined from the decrease in chromaticity.

A polarographic method using a platinum electrode is known as an electrochemical method for measuring hydrogen peroxide. There is also a method of quantifying hydrogen peroxide by measuring the rise and rate of oxygen levels associated with oxygen production using an enzyme electrode that also uses catalase.

In addition, there is a method in which iodine anion is oxidized to iodine with hydrogen peroxide in the presence of a molybdate catalyst, and the production rate is detected and quantified with a potentiometer or an ammeter.

As a quantitative determination method for hydrogen peroxide, homovanillic acid was 2,2'-
Dihydroxy-8,8'-dimethoxy-diphenyl-5
, 5'-diacetylic acid is converted into a phosphor and measured. In addition to homovanillic acid, substances that are converted into fluorescent substances by hydrogen peroxide and peroxidase include p-hydroxyphenylacetic acid and diacetyl-
Examples include 7'-dichlorofluorescein. In addition, fluorescent substances such as 6-medquine-7-hydroxy-1,2-benzopyrone and 3,5-diacetyl-1,4-dino-1-hydroltidine are oxidized to non-fluorescent substances by hydrogen peroxide-,<-oxidase. There is a method of converting the light and measuring the decrease of one light.

Removal of the influence of interfering substances In the indicator substance detection system as described above, substances other than the indicator substance in the object of quantification may interfere with detection.

In such cases, it is necessary to eliminate the influence of interfering substances.

For example, in an enzymatic reaction at pH 7.0 to 8.0,
SH compounds other than GSH and L-Cys in the quantitative target substance or in enzymatic reactions at pH 4.5 to 5.5, SH compounds other than GSH remain as they are even after GSO is applied, but if SH compounds are present, Color development by the color reagent described above is inhibited, interfering with L-Cys measurement. In such cases, it is necessary to block the SH compound with an SH group blocking agent.

As the SH group blocking agent, N-ethylmaleimide (hereinafter referred to as
It is abbreviated as NEM. ) is preferred).

Hereinafter, an example of implementation of the method of the present invention will be shown and a specific explanation will be given. However, these I! This is merely an example and is not intended to limit the method of the present invention. In addition, G S 01! used in each example. According to the method of the previous example for reference #≠ Cloud chewing penis 1 noum acreatum IFO
It was prepared from a culture of No. 5729.

Example coloring reagent: Phenol 20117, 4-aminoantipyrine 12■ and horseradish, <-oxidase (manufactured by Toyobo ■, grade l) 2.5■ (100tJ/mη)
Some's 0.2M potassium phosphate buffer (pH 7,
4).

GSO solution 1: 50μq of purified enzyme (540U/W)
4N/0.02M acetate buffer (+)H5,O) was dissolved ('0.5 U/gl).

GSO solution 2: Purified enzyme (540U/+y) 50Mg
of 5.4 ml of 0.02M potassium phosphate buffer (1
) H7,4) (5.0 U/ml).

NEM solution: 101 µmol of NEM 200 μmol!
1 ml of 0.5M potassium phosphate buffer (1)H7,4)
dissolved in

Procedure: Put 0.1 ml of 0.5 M acetate buffer (pH 5,0) and 0.1 ml of G5-0 solution 1 into a test tube (A), of 0.5M potassium phosphate buffer (+)H7,4) and 0.5M potassium phosphate buffer (+)H7,4). i ml
of GSO solution 2 and placed in a constant temperature bath at 37°C. Test tubes (a) and (b) each k lc L −
cys (0-0.8 μmol/*/) GSH (0,471
The reaction was started by adding 0.5 #l/ of a standard solution containing mol/*/), and after incubation with aerobic shaking for 20 minutes, 0.1 t of NEM solution was added to each test tube.
el was added, followed by 0.2 ml of coloring reagent.

Measure the absorbance at 500 nm (B'# and A'; amounts proportional to the amount of hydrogen peroxide produced) of each of the test tubes (a) and (b) with no sample added as a control, and calculate the difference t between the constant values on both sides.
xg) and created a calibration curve (Figure 1).

[Brief explanation of the drawing]

FIG. 1 shows a calibration curve in Examples. In the figure, the vertical axis is the OD at pH 7.4. Absorbance at 500 nm (difference from lees (A'-B') is shown, and the horizontal axis shows the amount of L-Cys (μmol/tube)
shows.

Claims (1)

[Claims] 1) When quantifying cysteine in a sample, glutathione sulfhydryl oxidase is applied to the sample in the presence of oxygen, and the amount of oxygen consumed or the amount of reactant produced accompanying the reaction is measured. A method for quantifying cysteine, which is characterized by: 2) Enzyme reaction by glutathione sulfhydryl oxidase at pH 7.0-80 and pH 4.5-5.
．． 5, and the oxygen consumption amount +A+ or the amount of reactant production +A+ in the reaction solution at pH 7.0 to 8.0, and p
The amount of oxygen consumed in the reaction solution (Bl or amount of reactant produced 1B') in H4,5-55 was measured, and the difference between them (A
The method for quantifying L-cysteine according to claim 1, in which the L-cysteine content is determined from -B or R-B:). 3) The method for quantifying cysteine according to claim 1, wherein the sample is a sample substantially free of glutathione. 4) The method for quantifying cysteine according to any one of claims 1 to 3, in which the amount of produced hydrogen peroxide is used as the amount of produced reactant to be measured.