SU1359253A1 - Method of spectrometric determination of amidase activity of enzymes detectable group for performing same - Google Patents

Abstract

The invention relates to the field of bioorganic chemistry, in particular, to methods for analyzing enzymes that hydrolyze amide bonds. The invention can be used in food, photo, microbiological industry, medicine and agriculture. The aim of the invention is to accelerate and simplify the process. To determine the amidase enzymatic activity using 1- (aminoacyl) amino naphthalene-5-sulfonic acids, the latter are introduced into the reaction mixture containing the buffer solution, other components necessary for the enzyme activity (mercaptoethanol, complexones , inorganic ions, etc.), the mixture is thermostated, the enzyme is added and the change in optical density is measured either in the absorption region of the product (nm) or in the absorption region of the substrate h (nm). The first is preferable, since other compounds usually found in the mixture do not absorb in this area. 1- (aminoatsh1) aminonaphthalene-5-sulfonic acid (AA) under the action of the enzyme removes 1-amino naphthalene-5-sulfonic acid (ANSC): RTS1H 2 (O1O) ROH + 280 nm) or increment ANSC (330 nm). E-trypsin, thrombin, papain, pronaza K-leucyl, tosylglycylprolylarginyl, carbobenzoxy-arginine, 2 s, and 3 s. f-ly, 2 tabLo with (L

Description

This invention relates to bioorganic chemistry, and specifically to methods

analysis of enzymes that hydrolyze amide bonds.

The invention can be used in food, photography, microbiological industry, medicine and agriculture.

The aim of the invention is to accelerate and simplify the process.

The method is as follows.

To determine the amidase enzyme activity using 1- (amy noadyl) aminonaphthalene-5 sulfonic acids, the latter are introduced into the reaction mixture containing a buffer solution that is optimal for the enzyme under study, other components necessary for the manifestation of enzymatic activity (mercaptoethanol, complexones, inorganic ions et al.), the mixture is thermally stabilized, the enzyme is added and the change in optical density is measured either in the absorption region of the product (330), because in the absorption region of the substrate (280 nm). The first is preferable, since other compounds normally found in the mixture do not absorb in this area.

Example 1. Determination of trypsin activity by decomposition of arbobenzoxyarginyl) aminonaphthalene-5-sulfonic acid.

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25

20

2 ml of the reaction mixture containing 5-10 M 1 N -carbobenzoxyargi35

lO- M

Nile) -aminonaphthalene-5-sulfonic acid in 0.05 M tris-HC1-buffer pH 8.2, placed in a spectrophotometer cuvette, thermostatic at 25 or 37% addition-40 synthesis; the presence of ZORN-groups, increased proteases, including blood proteases, and 1- (aminoacyl) amino-naphthalene-5-sulfonic acids (or their salts) are used as substrates for determining the activity of enzymes. Additional advantages of 1-aminonaphthalene-5-sulfonic acid as a chromophore group of enzyme substrates are intense fluorescence, which allows controlling the process at all stages.

chivayuschee solubility in aqueous solutions; the ability to azo coupling, both as azo and after preliminary diazotization, diazocomposition with the formation of intensely colored dyes and; availability (1-aminonaphthalene-5-sulfonic acid is used as a starting compound in the synthesis of dyes, its production is mastered by industry and is carried out on a significant scale).

50 µm of the enzyme under study are injected and the spectrum in the region of 400 ± 240 nm is recorded in the rescanning mode. The amount of enzyme activity is determined by the increase in absorption in the region of 330 nm or by the decrease in absorption in the region of 280 nm. Absolute values are determined by the molar extinction of 1-aminonaphthalene-5-sulfo acid, relative values are determined by a calibration curve constructed from a standard enzyme solution.

Example2. 2MP of the reaction mixture containing lecylaminonaphthalene-5-sulfonic acid bromide in 0.05 M tr is-HCl buffer pH 7.6, incubated at 25 ° C, added

10 µl of the study drug - Pro nazy (a mixture of proteinases used

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in biochemical studies), incubated for 10 min., add 1 ml of 0.1 M NaOH solution, measure the optical density at 330 nm relative. the solution to which the enzyme is added immediately prior to measurement. The results for examples 3-6 are given in table. one.

Acceleration and simplification of the process is achieved by eliminating the extraction and centrifuging operations, due to the fact that it is used as a detectable group 1-a: min5 naphthalene-5-sulfonic acid soluble in aqueous solutions, and unlike 2- (aminoacyl) - The 1-naphthylamines used in the prototype are not carcinogenic and readily available. The advantage of the proposed method over the prototype is given in table. 2 and 3.

As a detectable group, 1-aminonaphthalene-5-sulfo5 acid, which is not carcinogenic, is proposed to be sufficiently soluble in aqueous solutions, especially, i.e. in the area of maximum activity of many

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synthesis; the presence of ZORN-groups, increased proteases, including blood proteases, and 1- (aminoacyl) amino-naphthalene-5-sulfonic acids (or their soybeans) are used as substrates for determining the activity of enzymes. Additional advantages of 1-aminonaphthalene-5-sulfonic acid as a chromophore group of enzyme substrates are intense fluorescence, which allows controlling the process at all stages.

synthesis; the presence of ZORN-group, increasing the solubility in aqueous solutions; the ability to azo coupling, both as azo and after preliminary diazotization, diazocomposition with the formation of intensely colored dyes and; availability (1-aminonaphthalene-5-sulfonic acid is used as a starting compound in the synthesis of dyes, its production is mastered by industry and is carried out on a significant scale).

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As a result of the refusal to use carcinogenic substances - naphthylamine production, it is possible to produce substrates for enzymatic analysis by the industrial sector of the USSR (expanding production capacity), there is no need to import substrates from abroad, improve

Claims (1)

under the conditions of the personnel, as a result of where R is the amino acid residue, the increase in solubility increases, the maximum allowable concentration in the sample, which allows the analysis of enzymes with low affinity to the substrate (expansion of the study area), and also provides the possibility of analyzing enzymes at low temperatures. Invention Formula 1. A method for the spectrometric determination of the enzyme amidase activity, comprising: 3. The method according to claim 1, characterized in that 1 (tosylglycyl-1 enzymatic hydrolysis reaction of amy-, 5 Prol1-1-arginyl) aminonaphthalene-5ioacyl derivative of naphthalene row is used as a substrate; In order to speed up and simplify the process, 1- (aminoacyl) aminonaphthalen-5-sulfo acid of general formula: RNH is used as an enzyme substrate. sulfonic acid or its salt. 4. The method according to claim 1, characterized in that the reaction product is determined spectrophotometrically at -L 330 nm and in the same solution where the enzymatic reaction was carried out, 5, Use of 1-aminonaphthalene-5 25 sulfonic acid as a detectable group of enzyme substrates with amidase activity. amino acids or peptide, or their salts. differ2 The method according to claim 1, yi and the fact that 1- (N -carbobenzo-arginyl) aminonaphthalene-5-sulfonic acid or its salt is used as a substrate. 3. A method according to claim 1, characterized in that 1 (tosylglycyl-1-prol-1-1-arginyl) aminonaphthalene-5 sulfonic acid or its salt is used as a substrate. 4. The method according to claim 1, characterized in that the reaction product is determined spectrophotometrically at -L 330 nm and in the same solution where the enzymatic reaction was carried out, 5, Application of 1-aminonaphthalene-5-sulfonic acid as a detectable group of substrates of enzymes with amidase activity. Trypsin 1-Tozsh1Glycyl-1-prolyl-1 arginyl) aminonaphthalene-5-sulfonic acid Thrombin Same Papain 1- (1-N -carbobenzoxyarginyl) aminonaphthalene-5-sulfonic acid Thrombin Same Table 1 For example, 1 10 25 2 37 10 25 30 37 Table 2 Indicator Method Prototype Proposed Incubation Time 2 h 2-30 min DiazolIs No Ethyl acetate Extraction Stage Centrifuging step there is no transfer operation3 o T a-b l and c a 3 Indicator By prototype Detectable group CarcinogenicityIs No AvailabilityManufacture Produced in the USSR banned in the USSR Ability to educate water soluble dye in the azo coupling reaction there is no Compiled by L. Borisova Editor N. Gorvat Tehred M. Khodanych Proofreader A, Obruchar Order 6109/23 Circulation 776 Subscription VNIIPI USSR State Committee for inventions and discoveries 113035, Moscow, Zh-35, Raushsk nab., 4/5 Production and printing company, Uzhgorod, Projecto st., 1