![]() Method of identifying hydrogen peroxide in fermentation reaction
专利摘要:
The invention relates to biological chemistry, more specifically to the determination of hydrogen peroxide during an enzymatic reaction. The purpose of the invention is to increase the reproducibility of the method by maintaining the stability of the colored complex. In this test, an oxidase buffer, an antipyrine derivative and a phenolic compound are added to the test sample. The level of hydrogen peroxide of the proceeding enzymatic reaction is determined photometrically. As a phenolic compound combined with 4-aminoantipyrine, p-chlorophenol, phenol and other derivatives thereof, aniline derivatives, naphthol and its derivatives, naphthalinamine and its derivatives, aminoquinoline, oxyquinolines, and the like are optimal. The reaction is carried out in a buffer solution with a pH of 6-9. 1 tab. I O) with from 00 00 cm 公开号:SU1338787A3 申请号:SU813244903 申请日:1981-02-04 公开日:1987-09-15 发明作者:Георг Батц Ханс 申请人:Берингер Маннхайм Гмбх (Фирма); IPC主号:
专利说明:
1133 The invention relates to biological chemistry, in particular to the determination of hydrogen peroxide during an enzymatic reaction. The aim of the invention is to increase the reproducibility of the method by maintaining the stability of the colored complex. The method is carried out as follows. A buffer oxidase solution, an antipyrine derivative, and a phenolic compound are added to the test sample. Using photometry of the obtained colored complex, the level of hydrogen peroxide is determined, which forms during the enzymatic reaction. According to the invention, conventional phenolic compounds can be used, which can be combined with 4-aminoantipyrine to form a dye. Preferred as the phenolic compound is p-chloro) phenol. Examples of other suitable compounds are phenol, other derivatives of phenol, aniline derivatives, naphthol, naphthol derivatives, naphthalene-amine, naphthalinamine derivatives, amino quinolines, hydroxyquinolines, dioxyphenylacetic acid, and the like. The reaction is carried out in a buffer solution. Conditions known in peroxidase chemistry are suitable as buffers and pH values. The preferred pH values of the medium are in the range of 6 to 9. Otherwise, the choice of buffer and the pH value in the case of a reaction involving an enzymatic formation are determined primarily by the requirements placed on the buffer and the pH value of the enzyme involved in the reaction. Example 1. Determination of the formation of HjOj during the oxidation of glycerol using glycerol oxidase. . A reagent of 1: 0.1 mol / trizthanololamine buffer (HC1, pH 8.0, 3.6 mmol / l or 2 g / l isotridecyl ester, 4.7 mmol / l or 2 g / l sodium chloride is prepared. , 10 mmol / l p-chlorophenol, 0.5 mmol / l amino-substituted 4-amino-antipyrin, 10 U / ml peroxidase); reagent 2: 500 U / ml glycerol oxidase .. To determine this, 2 ml of reagent 1 and 0.2 ml of reagent 2 are added with a pipette to the cuvette. The extinction EI, counted on the potentiometer at 546 them. Immediately after that The reaction is started by adding 20 ml of the sample. After 20 minutes, the yield is measured. E. The incubation temperature is. The evaluation is carried out through a calibration straight line, at which the measured difference of extinction (1 U E / - - E), the standard solution of glycerol to the concentration of glycerol is in the ratio. The concentration of the test mixture was 0.09 mol / l of triethanolamine HCf buffer; pH 8.0, 1.8 g / l (3.2 mmol / l) isotridecyl ester, 4.3 mmol / l sodium cholate, 9 mmol / l p-chlorophenol, 0.45 mmol / l amino-substituted 4-aminoantipyrine, 4 U / ml peroxidase, 45 U / ml glycerol oxidase. Example 2. The color stability of five compounds was investigated. As compounds for comparison, 4-aminoantipyrine and sulfated 4-aminoantipyrine are taken. With these comparative compounds and novel compounds, using p-chlorophenol, 2,4-dichlorophenol and ethyloxytoluidine (sulfonated) as the phenolic component of the combination, EXTINCTION f was determined, the stability of the resulting dye and The following reagents were used during the test: reagent 1: O, 1 mol / l potassium phosphate as buffer, pH 8.0, 0.1 mmol / l derived from 4-aminoantipyrine, 1.0 mmol / l phenolic component, 2 U / ml POD; reagent 2: 0.01 mol / l solution. Determining conditions: measuring radiation of 546 nm, layer thickness of 1 cm, incubation temperature at room. 2.0 ml of reagent 1 is pipetted into the cuvette, 10 µl of reagent 2 (sample) is added and mixed. The extinction is carried out after 70 minutes. Next, a control value is determined. In this case, instead of a solution, buffer is added to reagent 1. The research results are summarized in the table. The percentage stability data of the dye (60 minutes at room temperature) refers to the deviation relating to the measurement signal that occurs during the appearance. The table shows that the ox compounds of the invention have only a minor effect, also on the value of extinction. The compounds according to the inventive method are superior to the comparative compounds in terms of the stability of the colored complex.
权利要求:
Claims (1) [1] Invention Formula The method for determining hydrogen peroxide in an enzymatic reaction, including the interaction of a sample with a buffer solution of oxidase, the addition of an antipyrine derivative and a phenolic compound, followed by photometry of the colored complex, is distinguished by yen in that , µmol / ml 500 502 505 IO + 2Z 27 + IX 505 507 33 + 16 / J 32-1 1Z 36-21 Max 550 546550 Editor E. Kopcha Order 4151/59 Tehred I.Popovich Cor Podp Circulation 776 VNIIPI USSR State Committee for inventions and discoveries 113035, Moscow, Zh-35, Raushsk nab., 4/5 Production and printing company, Uzhgorod, st. Project, 4 In order to increase the reproducibility of the method by maintaining the stability of the colored complex, a substance selected from the group 4-aminoantipyrine, 1- (C-ethylaminophenol-4-aminopyrazolone- (5), 1- (amino-n- amino tolyl) -2, 3-dimethyl-4-aminopyrazolone- (5), 1- (p-diethyl-aminophenyl) -2, 3-dimethyl-4-aminopyrazolone- (5), in the amount of O , 1-0, 5 mmol / l of the reaction mixture. I9t0120tOZI9 IZ 503 502 503 25-IZ 22 + OZl5 + 2t 41 + lZ 508 507 508 502 23-3Z 25-7125-5Z37-1 548 546 550 546 Proofreader V. But ha ha Subscription
类似技术:
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同族专利:
公开号 | 公开日 DD156089A5|1982-07-28| YU28981A|1983-10-31| AU6693681A|1981-08-13| CA1154781A|1983-10-04| AU521113B2|1982-03-18| US4567139A|1986-01-28| US4394512A|1983-07-19| DE3160027D1|1983-02-24| AR226091A1|1982-05-31| EP0033539B1|1983-01-19| EP0033539A1|1981-08-12|
引用文献:
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申请号 | 申请日 | 专利标题 DE3004129|1980-02-05| DE3039207|1980-10-07| DE19813100807|DE3100807A1|1980-02-05|1981-01-13|Novel aminoantipyrine compounds and their use| 相关专利
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