SU873121A1 - Hydrolasa activity determination method - Google Patents

Description

(54) METHOD FOR DETERMINING; 1 GENGOLASE ACTIVITY

The invention othosit to determine the activity of hydrolyzing enzymes (hydrolases), and can be applied in biochemistry and in medicine to determine the activity of these enzymes. A spectrophotometric or fluorescent method for determining the activity of hydrolyzing enzymes is known, which consists in registering the rate of formation of chromophore or fluorophore products 1. However, this method is not applicable to opaque media, and moreover requires expensive equipment. The closest to the present invention is a method for determining the activity of a hydrolase, in particular, alkaline phosphatase, which consists in the interaction in the buffer solution of the enzyme with the substrate, which is an organic phosphoric acid ester, and the amount of free phosphate released during the reaction in the presence of specific j) eareHTOB. The spectrophotometric data obtained — the magnitude of optical density — judged the activity of enzyme C J. The disadvantage of this method is the impossibility of determining activity in opaque and colored media, and also that it includes an additional procedure for spectrophotometric determination of the reaction product in the presence of additional reagents. This method is not amenable to automation. The object of the invention is to simplify the process and make it possible to determine activity in opaque and colored environments. The goal is achieved by the method of determining the activity of hydrolases, consisting in the interaction of the enzyme with the substrate, which is organic, which is formed as a result of the reaction of pyrocatechin ion or p-aminophenol t ion, and determining the amount of the latter in the presence of an electrochemical converter at an anode potential of 0.18-0 , 3 V by measuring current. By the rate of current increase, the activity of the enzyme is judged. 38 Usually alkaline phosphatase or (J-glucosidase is used as hydrolases, in the first case pyrocatechin mono-orthophosphate or p-aminophenol mono-orthophosphate 0.18-2 mm is usually used as a substrate, and 0.1-0.2 M as a buffer solution Tris-HC & Buffer containing 0.01-0.02 M MgS04; in the second case, p-aminophenol D-glucoside 6.18-3 mM is used as a substrate, and 0.1-0 is used as a buffer solution. , 2 M acetate buffer. As alkaline phosphatase, enzyme systems of intact E cells with AND (i.e. alkali phosphatase contained directly in the above-mentioned cells), while pyrocatechol mono-orthophosphate in an amount of 0.18–3 mM is used as a substrate, and 0.1 0.2 M Tris-HC1 buffer containing 0, 01-0.02 M MgSO4. Significant differences of the method are: the use of an ether as a substrate, which forms a pyrocatechin ion or p-aminophenol t ion as a result of the reaction, determining the amount of the latter in the presence of an electrochemical converter at 0.3 V by measuring current and also h about the activity of the enzyme is judged by shrastani current speed. The drawing shows an electrochemical converter. An electrochemical converter consisting of a platinum anode, a reference electrode and an auxiliary electrode is used for carrying out the method (see. Drawing). The anode potential is selected in the range of 0.18-0.3 V, depending on the specific system. The method includes the following operations: dissolving the substrate in a buffer solution, immersing an electrochemical converter in the solution, introducing a detectable hydrolase into the solution, applying voltage, measuring the current. Example. To determine the alkaline phosphatase activity, take 0.1 M Tris-HCE, 0.01 M MgSO4 buffer, which contains 2 mM pyrocatechin mono-orthophosphate or 1 mM p-aminophenol mono-orthophosphate, pH 8.0.25 ° C. An electrochemical converter is immersed in the solution and a sample of alkaline phosphatase is injected. If pyrocatechol mono-orthophosphate is used as a substrate, the potential of the anode ycta is 0.3 V relative to the Ag / AgCB electrode, if mono-orthophosphate is used as the substrate, p-aminophenol, the potential of the anode is set to 0, 18 V relative to the Ag / AgCE electrode. Register an increase in current over time. The rate of current increase is directly proportional to hydrolase activity. The measurement takes 1-3 minutes (see Tables 1 and 2). The measurement of alkaline phosphatase activity in the presence of pyrocatechol mono-orthophosphate as a substrate is given in Table. 1. Measurement of alkaline phosphatase activity in the presence of p-aminophenol mono-orthophosphate as a substrate is given in Table. 2.. P and M e p 2. To determine the activity of | 3-glucosidase, take 0.1 M acetate buffer, which contains 1.5 mM D-O-glucoside p-aminophenol pH - 4.7, temperature 25 ° C. In solution immerse the electrochemical transducer and inject a sample of the detected (3-glycosidase. Anode potential is set to 0.3 V. relative to the Ag / AgCE electrode. An increase in current over time is recorded. The rate of increase in current is directly proportional to the hydrolase activity. The measurement takes 1-3 minutes. The measurement of the amount of | 3-glucosidase is given in Table 3. Regression analysis results This definition shows good agreement between the results of the proposed method and the known spectrophotometric methods, the correlation coefficients for the examples given in Tables 1, 2 and 3 are equal to 0.9998, 0.9989, 0.9999, respectively. To determine the alkaline phosphatase activity directly in intact E.coEi cells, take 0.1 M Tris-HCC 0.01 M IVlgS04 buffer containing 2 mM pyrocatechin mono-orthophosphate, pH 8.0, at 25 ° C. Immerse the electrochemical converter in the solution. and a sample of detectable hydrolase is introduced. The anode potential is set to 0.3 V relative to the Ag / AgCe electrode. . Register an increase in current over time. The rate of current quivering is directly proportional to hydrolase activity. Measurement takes 1-3 minutes. The determination of alkaline phosphatase activity directly in intact E.O. coBi cells is given in Table. 4. From table. 4, it can be seen that there is a strictly separate relationship between the number of cells and the enzyme activity detected. Example 4. The dependence of the rate of increase in the current of the converter on the concentration of enzymes is described by the Michaelis dependence. The dependence of the rate of increase in current on the concentration of substrates is established at an alkaline phosphatase concentration of 0.026 units / ml, pH 8.0 in both substrates, in 0.1 M Tris-He, 0.01 M MgSO4.25 ° C. The potential of the anode for the mono-orthophosphate of p-aminophyllum is 0.18 V relative to the Ag / AgC electrode. The concentration of / 3-glucose on p-aminophenol | 3-0-glucoside is 0., pH 4.7, in 0.1 M acetate bu 58731256

Phere, at 25 ° C. Anode potential of 0.3 V; relative current increase versus substrate concentration

Ag / AgCE electrode. The dependence of the rate in the presence of hydrolases is given in table. five.

Converter current change rate, nA / min

3,662,6

5.05.2

6,717,8

8,010,4

11,613,0

13.0 .15.6

12.6618.2

19.33., 23.4

The current change is the amount of alkaline phosphachase,

transformer, nA / min I10 units / ml

..

52.6

2.3 -7.8

20.7. 13.0

39.3 23.4

The rate of change of currentNumber | 3-glucosidase,

the Converter, nA / min10 u / ml

0.73

1.53

2.6

3.4

6.5

Table 1

The amount of alkaline phosphatase,

0 u / ml

Table 2

Table3

3.7

7.4

14.5

22.3

37.0

Speed change current converter, nA / min

1.47

2.87

4.5

5.95

7.3

Claims (4)

1. A method for determining the activity of hydrolases, including the interaction of the enzyme with a substrate, which is an organic ester, in a buffer solution and determining the amount of products formed during the reaction, characterized in that, in order to simplify the process and enable the determination of activity in opaque and colored mediums, esters are used as substrates, which form a pyrocatechin-ion or p-aminophenol t-ion as a result of the reaction, the latter are quantitatively determined in the presence of electrons At a potential of the anode of 0.18–0.3 V, a chemical converter determines the enzyme activity by measuring the current and the current rise rate. The number of cells in cm 4.6. ten 9,2- 10 13,810 18,410 2310 Table 5 2. The method according to claim 1, that is, that alkaline phosphatase is used as the hydrolase, pyrocatechin mono-orthophosphate or p-aminophenol mono-orthophosphate is used as a substrate, and 0.18-3 mM as a buffer. 0.1-0.2 M solution of Tris-NSC buffer containing 0.01-0.02 M MgS04 3. Method according to Ш1. 1 and 2, differing in those; that p-aminophenol (3-D-glucoside) is used as the substrate in the amount of OJ8-3 mM, and 0.1-0.2 M acetate buffer is used as the buffer solution. 4. A method according to claim 2, characterized in that alkaline phosphatase is used, which is contained directly in intact E. coli cells, while pyrocatechol mono-orthophosphate 0.18- is used as a substrate; 3 mM, and E as a buffer solution of OD-0, 2 M Tris-HCB buffer containing 0.010, 02 M MgS04.V Source of information taken into account in the examination 73115.Ш 1 H I, Rufgrneys, Methods of Enayrnatic AlaPzE8 AsKiee-is Prsss,, London, 1965, 783. 2. E (bi H, VViniam E.M. Lands, A nevy Bensitike Determination of Phosphate, AnaL 57 (prototype). BtecheiT: leSP.,;