JPH0218839B2 - - Google Patents

Description

[Detailed description of the invention]

[] Background of the Invention The present invention relates to a method for quantifying L-cysteine (hereinafter abbreviated as L-Cys) using a new enzyme, glutathione sulfhydryl oxidase (hereinafter abbreviated as GSO). . Conventionally, methods for quantifying L-Cys include the acid ninhydrin method or chemical methods using reagents that react with SH (thiol) groups such as 5,5'-dithiobis-(2-nitrobenzoic acid) and o-phthalaldehyde. However, no method has been established that is necessarily satisfactory in terms of specificity, simplicity, sensitivity, or influence of contaminants. The present inventor previously discovered that a new sulfhydryl oxidase, GSO, with high specificity for glutathione (hereinafter abbreviated as GSH) is produced by microorganisms, and The properties were clarified (see Japanese Patent Application No. 16993-1983). As a result of further research to apply this enzyme to the quantification of L-Cys, the inventor discovered that other enzymes such as GSH
Even if SH compounds coexist in the sample, L in the sample
-We have discovered that Cys can be quantified specifically, with high sensitivity, and easily by utilizing the change in specificity of GSO due to PH, and have completed the present invention. [] Summary of the Invention 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. This provides a method for quantifying L-Cys. Especially L-Cys in samples containing GSH
When quantifying the oxygen reaction caused by GSO, the PH
Performed at 7.0-8.0 and PH4.5-5.5, PH7.0-
Measure the oxygen consumption amount A or reactant production amount A′ in the reaction solution at 8.0, and the oxygen consumption amount B or reactant production amount B′ in the reaction solution at PH 4.5 to 5.5,
From their difference (A-B or A'-B'), L-Cys
It provides a method for determining the content. Effects GSO used in the present invention has high specificity for GSH at pH 4.5 to 5.5, and if the optimal enzyme amount is used, it will reduce the SH compounds present in the sample.
Oxidizes only GSH almost specifically, but at pH 7.0 to 8.0
When sufficient enzyme is present, GSH
At the same time, L-Cys can also be completely oxidized. Therefore, as in biological samples, there are
In the case of a sample in which most of the SH compounds are occupied by GSH and L-Cys, from the oxygen consumption amount A or the reactant production amount A' at PH7.0 to 8.0, PH4.5
Oxygen consumption B or reactant production at ~5.5
By subtracting B', only the amount of L-Cys in the sample can be determined easily and accurately. Note that the sample contains SH compounds other than L-Cys, especially
PH7.0-8.0 when GSH is virtually absent
It goes without saying that L-Cys can be quantified only by measurement at . [] Detailed Description of the Invention 1) Enzyme-chemical and physico-chemical properties of glutathione sulfhydryl oxidase The enzyme-chemical and physico-chemical properties of the purified enzyme preparation of GSO in the present invention are as follows. (1) Effect GSO is a known sulfhydryl oxidase from rat seminal vesicle secretion (Biochemistry 19, 2639-2645)
(1980)), two molecules of SH compounds are oxidatively condensed in the presence of oxygen to produce the corresponding disulfide compounds, but especially
Shows strong affinity and high substrate specificity for GSH,
It is a novel sulfhydryl oxidase that exhibits only weak activity against SH groups in proteins. When GSH is used as a substrate, GSO requires 1 mol of oxygen per 2 mol of GSH, and oxidized glutathione and 1 mol of oxidized glutathione, as shown in the reaction formula below.
Generates 1 mol of hydrogen peroxide. Also, when L-Cys is used as a substrate, GSH
As in the case of , for every 2 mol of L-Cys,
Requires 1 mol of oxygen, 1 mol of cystine and
Generates 1 mol of hydrogen peroxide. 2RSH＋O ₂ →RSSR＋H ₂ O ₂ (In the formula, RSH is GSH or L-Cys,
RSSR indicates oxidized glutathione or cystine. ) (2) Substrate specificity GSO purified for various SH compounds
The results of the reaction at acidic (PH5.0; 0.2M acetate buffer), neutral (PH7.4; 0.2M phosphate buffer), and alkaline (PH9.3; 0.2M Tris buffer) This is Table 1. In Table 1, the concentration of each substrate is 5mM. In addition, the enzyme activity was measured by the oxygen electrode method described below, and expressed as a relative value of the activity against GSH at each PH.

[Table] As is clear from Table 1, GSO showed high substrate specificity for GSH at pH 5.0. PH
In addition to GSH, 7.4 also acted efficiently on L-Cys and dithiothreitol;
When acting on a biological sample, since dithiothreitol is substantially absent, L-Cys as well as GSH can be completely oxidized if a sufficient amount of enzyme is used. At PH7.4
As a result of measuring the Km value, the Km value is 6.8×10 ^-4 M for GSH, and Km value for L-Cys.
The value was 3.7×10 ^-3 M. GSO is a sulfhydryl compound from rat seminal vesicle secretion that has high substrate specificity and strong affinity for dithiothreitol.
It is clearly distinguished from oxidase. Furthermore, GSO hardly acts on the SH groups of proteins, and in this respect it could be distinguished from known enzymes. For example, the SH group of ribonuclease A (RNase A; molecular weight 13,700) reduced in 2-mercaptoethanol in the presence of 8M urea.
When GSO was applied, even when a sufficient amount (5U/ml) of GSO was added,
The rate of decrease in SH groups (8 groups) per molecule of RNase A was approximately 1.5 per 4 hours, which was slightly higher than the rate of decrease in SH groups due to natural oxidation (1.0 groups/4 hours). On the contrary,
Known rat seminal vesicle secretion sulfhydryl
When oxidase is added at 0.5U/ml,
It shows a decrease of 7.5 molecules/mol or more in 4 hours (see Fig. 7 of the aforementioned Biochemistry 19, 2639 (1980)), and is an enzyme that exhibits remarkable activity against RNase A. That is, the GSO of the present invention is a novel flavoprotein sulfhydryl oxidase that exhibits strong affinity for GSH under acidic and neutral conditions and acts specifically on it. (3) Measurement of titer The titer of GSO was measured using an oxygen electrode method. That is, 1 ml of 0.1M potassium phosphate buffer (PH7.4) containing 10mM GSH was placed in an oxygen electrode cell, 10μ of oxygen solution was added, and the oxygen consumption rate was measured. for 1 minute at 30℃
The amount of enzyme that consumes 1 μmol of oxygen is 1 unit (Unit; herein abbreviated as U).
And so. (4) Optimal PH The optimal PH is around PH6.5-7.7, especially PH7.1-7.8
It's nearby. The optimum PH can be measured using 0.15M sodium acetate-hydrochloric acid buffer, 0.15M sodium acetate-acetic acid buffer, 0.15M potassium phosphate buffer, 0.15M Tris-hydrochloric acid buffer and 0.15M
Using glycine-sodium chloride-sodium hydroxide buffer, PH2.4-10.8
I went within the range of (5) PH stability and thermal stability PH stability is determined from PH3.0 to
10.8, the enzyme activity was measured after holding at 45°C for 15 minutes, and it was found to be stable in the pH range of 5 to 9. In addition, thermal stability was determined by holding the enzyme at 40 to 80°C for 15 minutes in 0.1M potassium phosphate buffer (PH7.4), and measuring enzyme activity. The result is 55
It remained fully active up to 80°C.
It was completely deactivated. (6) Range of suitable temperature for action In the oxygen electrode method used to measure the enzymatic activity of GSO, the amount of dissolved oxygen in the reaction solution varies depending on the temperature, so the exact range cannot be specified, but the production of hydrogen peroxide is When measured using a known method, a temperature range of about 30 to 50°C was suitable. (7) Inhibition, activation and stabilization Potassium cyanide, calcium chloride, potassium chloride, nickel chloride, aluminum chloride, strontium chloride, barium chloride, ferric chloride, magnesium sulfate, zinc sulfate, manganese sulfate, copper sulfate, lithium sulfate, iodine Enzyme reactions were performed in 0.1M potassium phosphate buffer (PH7.4) containing various metal salts such as potassium chloride and 1mM of inhibitors such as ethylenediaminetetraacetic acid (EDTA), and the presence or absence of inhibition by each substance was investigated. As a result, the activity of GSO was completely inhibited by zinc sulfate, that is, zinc ions, and about 20% by manganese sulfate, but was hardly inhibited by other inhibitors. (8) Ultraviolet absorption spectrum λ _nax 272nm, 365nm, 443nm E280nm 1% 14.78 (9) Coenzyme The supernatant obtained by heat treatment or trichloroacetic acid (TCA) treatment and centrifugation is
Since its absorption spectrum matched that of flavin adenine dinucleotide (FAD) and it activated the apoenzyme of D-amino acid oxidase, it was revealed that the coenzyme of this enzyme was FAD. Also, FAD is per mol of GSO
2mol is present. (10) Polyacrylamide gel electrophoresis and
SDS-polyacrylamide gel electrophoresis The purified enzyme showed a single band by both methods. (11) Isoelectric point Ampholine (Sweden, LKB
The pI was 4.21 when measured by isoelectric focusing using an electrophoresis method (manufactured by Phys. Co., Ltd.). (12) Molecular weight The molecular weight of GSO is Sephadex G-200.
(manufactured by Pharmacia Huain Chemicals)
It was determined to be 94000±5000 by gel filtration method. In addition, by SDS-polyacrylamide gel electrophoresis, GSO has a molecular weight of 47000±
It was shown to be a dimeric enzyme composed of two subunits of 3000. (13) Crystal structure and elemental analysis GSO is not crystallized and therefore not measured. (14) Purification method GSO can be purified by appropriately combining salting-out method, isoelectric precipitation method, precipitation method using an organic solvent, adsorption method using diatomaceous earth, activated carbon, etc., various chromatography methods, etc. 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 the production method, a method for producing GSO using microorganisms will be specifically shown as a reference example. Reference example Put 8 g of wheat bran, 5 ml of water, and 1 g of rice husks into a 300 ml Erlenmeyer flask and autoclave at 120°C for 30 minutes.
Aculatum IFO 5729 was inoculated and cultured at 28°C for 7 days to prepare an inoculum. 200 5-volume Erlenmeyer flasks with 200 bran each
g and 140 ml of water, autoclaved at 120℃ for 30 minutes, inoculated with the above-mentioned seed culture, and incubated at 28℃ for 7 minutes.
Cultured for 1 day. The obtained culture was immersed in 30 g of water for 1 hour, filtered, and further passed through diatomaceous earth to obtain about 2 g of crude enzyme solution. Ammonium sulfate was added to this crude enzyme solution up to 65%, and the generated insoluble matter was removed by centrifugation. Ammonium sulfate was further added to the supernatant to achieve a 95% saturation concentration, and the resulting precipitate was collected by centrifugation and diluted with 0.02M phosphate buffer (PH7.4) 1
and dialyzed against the same buffer overnight. DEAE (diethylaminoethyl) - the precipitate generated during dialysis was removed by centrifugation, and the supernatant was equilibrated with the same buffer.
It was passed through a cellulose column (5 x 70 cm) and the adsorbed enzyme was eluted using the same buffer containing 0.2M NaCl. The eluted active fraction was collected, and after dialysis and concentration,
Gel filtration was performed using a Cephadex G-100 (manufactured by Pharmacia Fine Chemicals) column (3.5 x 100 cm), and the active fraction was collected and concentrated.
Dialysis was performed with 0.02M potassium phosphate buffer (PH7.4). This dialysis fluid was centrifuged, the supernatant fluid was microfiltered, and then lyophilized to produce a purified GSO specimen (yield 41%,
40 mg of specific activity 540 U/mg protein was obtained. Microorganisms used The microorganisms used in the production of GSO of the present invention are:
Aspergillus (hereinafter abbreviated as A.) genus, Penicillium (hereinafter abbreviated as P.) genus, Fusarium (hereinafter abbreviated as F.) genus, Trichoderma (hereinafter abbreviated as F.), Trichoderma (hereinafter abbreviated as F.) , abbreviated as T.), genus Paecilomyces;
It is abbreviated as pae. ) or Gliocladium (hereinafter abbreviated as G.), and is a microorganism that has GSO-producing ability.
A specific example of GSO-producing bacteria belonging to these genera is Aspergillus awamori (A.awamori).
IAM 2299, Aspergillus batatae (A.
batatae) IAM 2098, A.inuii IAM 2255, A.ochraceus IFO 4345, A.oryzae IFO
4183, Penicillium acreatum (P.
aculeatum) IFO 5729, Penicillium jenseni (P. jenseni) IAM 7197, Penicillium
P.citrinum Thom 1131
1131) Microtechnology Research Institute No. 1363, Fusarium solani (F. solani) IFO 5232, Fusarium sambucinum (F. sambucinum) OUT 4020, Gliocladium deliquescens (G.
deliquecens) OUT 4616, Paecilomyces varioti (Pae.varioti) IFO 4855, and Trichoderma viride (T.viride) ATCC 20536. However, the microorganisms used in the present invention are not limited to these strains. In addition, these GSO-producing bacteria can be mutated by conventional microbial mutagenesis methods, such as physical treatments such as ultraviolet rays, X-rays, and γ-ray irradiation, and chemical treatments with drugs such as nitrosoguanidine. Any of the high GSO-producing mutant strains obtained 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 this enzyme and the type of subsequent detection system for the indicator substance. That is, as long as it does not contain substances that interfere with the oxidation reaction by this enzyme and the reactions and/or detection in the indicator substance detection system,
The presence of contaminants in GSO enzyme preparations is not a problem. Furthermore, depending on the implementation 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 involves the oxidation reaction of L-Cys in a sample by GSO, especially GSH
When dealing with coexisting samples, it is necessary to measure the specific oxidation reaction at two points, PH4.5 to 5.5 and PH7.0 to 8.0, and the amount of oxygen consumed or the amount of reactants produced. It consists of a detection system for the reference material. Enzyme reaction using GSO The enzymatic reaction conditions for GSO for quantifying L-Cys are as follows. That is, the reaction PH is
Any PH range that is within the GSO stable PH range and that acts on L-Cys is sufficient, but especially when measuring L-Cys in samples coexisting with GSH, PH4.5 to 5.5 and PH7.0 to 8.0 are suitable. Must be in the PH range, both
Only by carrying out the reaction in the PH range
Changes in substrate specificity depending on the pH of GSO can be utilized. In the above case, the GSO of the filamentous fungus of the genus Penicillium shown in the reference example can suitably utilize the change in substrate specificity due to PH, especially by performing the reaction around PH 5.0 and around PH 7.4. In addition, when targeting samples in which GSH coexists,
In reactions at pH 7.0 to 8.0, the amount of enzyme used relative to the substrate is such that L-Cys and GSH are completely reacted, and in reactions at pH 4.5 to 5.5, the amount of enzyme used is in excess to the extent that it only acts on GSH. amount is preferred. Furthermore, in order to maintain the pH of the enzyme reaction, it is preferable to use various buffer solutions as the enzyme reaction medium. As the buffer solution, the above-mentioned PH
It is not particularly limited as long as it can maintain the range and does not inhibit the enzyme reaction. For example, commonly used buffers such as phosphate buffer, Tris-HCl buffer, acetate buffer, and citrate buffer are suitable. 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 SH compound, and hydrogen peroxide is preferred as the indicator substance of the present invention. Quantification of L-Cys is performed by measuring the change in the amount of 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 A or the amount of production of one of the reactants, such as hydrogen peroxide, accompanying the enzymatic reaction at pH 7.0 to 8.0 is calculated.
A' and the amount of oxygen consumed B' accompanying the enzymatic reaction at pH 4.5 to 5.5 or the amount B' produced of one of the same reactants as above are detected and measured, and the difference between the two measured values (A
-B or A'-B'), L-
Examples include methods for quantifying Cys. of course,
When targeting a sample that clearly does not substantially contain GSH, the pH at which L-Cys reacts sufficiently,
For example, an enzymatic reaction is performed at PH7.0 to 8.0,
L-Cys can be easily quantified by measuring the amount of oxygen consumed or the amount of reactant produced. 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. Typical methods for detecting and measuring each indicator substance are illustrated below, but the detection system for these indicator substances is not limited to known methods, and may also employ various methods to be further developed in the future. It is considered possible. Warburg's pressure detection method and oxygen electrode method are known as methods for measuring oxygen consumption (Biochemistry Experiment Course 5 Enzyme Research Methods (Part 1) Part 35
~52 pages, Tokyo Kagaku Dojin, 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. Examples of hydrogen peroxide activators include peroxidase from horseradish or sweet potato, as well as iodide,
Any one type of molybdate or a mixture thereof can be effectively used. Indicators include o-dianisidine, o-tolidine, o-toluidine, 2,6-dichloroindophenol, benzidine, 3,3'-5,5'-tetraalkylbenzidine (3,3'-dimethyl-5,5 ′-
diethylbenzidine, etc.), 4-methoxy-1
- In addition to naphthol, combination indicators can be used. An example of a combination indicator includes, but is not limited to, a combination of 4-aminoantipyrine and phenol. For example, instead of phenol, polyhydric alcohols or phenol derivatives such as 2,4-dichlorophenol, catechol, resorcinol, hydroquinone, cresol, guaiacol, pyrogallol, orcinol,
Furthermore, aniline derivatives such as aniline, dimethylaniline, and diethylaniline can be used. On the other hand, as a substitute for 4-aminoantipyrine, 4-aminophenazone, various 4-
Examples include aminopyrazolone derivatives, 3-methylbenzothiazolinone-hydrazone (MBTH) and its sulfonic acid derivatives.
In addition to the above, 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 of oxidizing alcohol using aldehyde dehydrogenase, the oxidative decolorization of indigo carmine using a copper ion-histamine system, and the method of quantitative determination based on the loss of chromaticity are also known. 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 method for quantifying hydrogen peroxide using a fluorescence method,
A method is known in which homovanillic acid is converted into a fluorophore of 2,2'-dihydroxy-3,3'-dimethoxy-diphenyl-5,5'-diacetylic acid using hydrogen peroxide and peroxidase. In addition to homovanillic acid, substances that can be converted into fluorescent substances by hydrogen peroxide and peroxidase include p-hydroxyphenylacetic acid and diacetyl-2',7'-dichlorofluorescein. In addition, fluorescent substances such as 6-methoxy-7-hydroxy-1,2-benzopyrone and 3,5-diacetyl-1,4-dihydroltidine are oxidized to non-fluorescent substances by hydrogen peroxide-peroxidase. There are ways to convert and measure the reduction in fluorescence. Removal of the influence of interfering substances In the indicator substance detection system as described above, substances other than the indicator substance in the quantitative target may interfere with detection. In such cases, it is necessary to eliminate the influence of interfering substances. For example, in an enzyme reaction at pH 7.0 to 8.0, substances other than GSH and L-Cys in the quantitative target
SH compounds or SH compounds other than GSH in enzymatic reactions at pH 4.5 to 5.5 remain as they are even after GSO acts, but the presence of SH compounds inhibits color development by the coloring reagent mentioned above, and L-Cys measurement interfere with In such a case
It is necessary to block the SH compound with an SH group blocking agent. As the SH group blocking agent, N-ethylmaleimide (hereinafter 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 are merely examples and do not limit the method of the present invention in any way. In addition, the GSO used in the example
Penicillium was prepared according to the method of the reference example above.
It was prepared from a culture of Aculatum IFO 5729. Example Coloring reagent: 20 mg of phenol, 12 mg of 4-aminoantipyrine, and 2.5 mg of horseradish peroxidase (manufactured by Toyobo Co., Ltd., grade) (100 U/mg)
was dissolved in 50 ml of 0.2M potassium phosphate buffer (PH7.4). GSO solution 1: 54 μg of purified enzyme (540 U/mg)
ml of 0.02M acetate buffer (PH5.0) (0.5U/ml). GSO solution 2: 50μg of purified enzyme (540U/mg) at 5.4
ml of 0.02M potassium phosphate buffer (PH7.4) (5.0U/ml). NEM solution: 200 μmol of NEM was dissolved in 10 ml of 0.5 M potassium phosphate buffer (PH7.4). Procedure: Put 0.1ml of 0.5M acetate buffer (PH5.0) and 0.1ml of GSO solution 1 in a test tube (A), add 0.1ml of 0.5M potassium phosphate buffer (PH5.0) to a test tube (B).
7.4) and 0.1ml of GSO solution 2, respectively.
It was placed in a constant temperature bath at 37°C. Add L-Cys (0 to 0.8 μmol/ml) alone or L- to each of test tubes (a) and (b).
Standard solution containing GSH (0.4μmol/ml) along with Cys
After starting the reaction by adding 0.5 ml and incubating for 20 minutes with aerobic shaking, 0.1 ml of NEM solution was added to each tube, followed by an additional 0.2 ml of color reagent. Test tube with no sample added as a control
(a) and (b) each absorbance at 500 nm (B′ and
A′; amount proportional to the amount of hydrogen peroxide produced) is measured,
The difference between both measured values (A'-B') was determined and a calibration curve was created (Figure 1).

[Brief explanation of drawings]

FIG. 1 shows a calibration curve in Examples. In the figure, the vertical axis is the absorbance A' at OD500nm at PH7.4 and the absorbance at OD500nm at PH5.1.
The difference from B'(A'-B') is shown, and the horizontal axis shows the amount of L-Cys (μmol/tube).

Claims (1)

[Claims] 1. When quantifying L-cysteine in a sample that does not substantially contain glutathione, in the presence of oxygen,
Glutathione sulfide oxidase is applied to the sample, the amount of oxygen consumed or the amount of reactant produced during the reaction is measured, and the amount of L in the sample is determined from the measured value.
-L characterized by determining cysteine content-
Method for quantifying cysteine. 2. The method for quantifying L-cysteine according to claim 1, in which the amount of hydrogen peroxide produced is used as the amount of reactant produced to be measured. 3. When quantifying L-cysteine in a sample containing glutathione, an enzymatic reaction using glutathione sulfhydryl oxidase is performed in the presence of oxygen at pH 7.0 to 8.0 and pH 4.5 to 5.5,
Measure the oxygen consumption amount A or reactant production amount A' in the reaction solution at PH7.0 to 8.0, and the oxygen consumption amount B or reactant production amount B' in the reaction solution at PH4.5 to 5.5, and measure them. A method for quantifying L-cysteine content, which comprises determining the L-cysteine content in a sample from the difference (A-B or A'-B'). 4. The method for quantifying L-cysteine according to claim 3, in which the amount of hydrogen peroxide produced is used as the amount of reactant produced to be measured.