SU1698786A1 - Method for determining superoxide dismutase activity in biologic materials - Google Patents

Description

one

(21) 4730891/14 (22) 22.08.89 (46) 12/15/91. Bul No. 46

(71) Minsk State Medical Institute

(72) G.N.Smel Nska and A.I.Balakleevsky (53) 612.015 (088.8)

(56) J.B.C., 1972. v. 247, Mg 10, p. 3170.

(54) METHOD FOR DETERMINING SUPER-SIDDISMUTASE ACTIVITY IN BIOLOGICAL MATERIAL

(57) The invention relates to biology and medicine and can be used to determine superoxide dismutase activity in experimental and clinical studies, as well as in evaluating the activity of commercial preparations of the enzyme. The purpose of the invention is to increase the sensitivity and specificity of the method, achieved by using 6-hydroxapamine as a substrate for autooxidation and desferrioxamine as a chelating agent. 4 il.

The invention relates to biology and medicine and can be used to determine the activity of superoxide dismutase in biological material in experimental and clinical studies, as well as in assessing the activity of commercial preparations of the enzyme.

The purpose of the invention is to increase the specificity of the sensitivity of the method.

The goal is achieved by determining the activity of superoxide dismutase by incubating the biological material containing it with b-oxyphosphine and desferrioxamine (Desferal) in phosphate buffer at pH 7.0 and 7.5, followed by photometry of the colored autooxidation product of 6-oxydophamine.

FIG. Figure 1 shows the curve of the degree of inhibition of auto-oxidation of 6-oksidofamina in the presence of desferioxin and the concentration of superoxide dismutase; in fig. 2 - kinetics of auto-oxidation of 6-oxyfamine in the presence of hemolysate superoxide dismutase in rat red blood cells; in fig. 3 — the effect of variable-valence metal ion bonding agents on the autooxidation kinetics of 6-oxydopamine; in fig. 4 - pH effect on rat erythrocyte hemolysate superoxide dismutase activity.

The method is carried out as follows.

A 0.1 M phosphate buffer solution, pH 7.0-7.5, is introduced into the sample, then the biological material containing superoxide dismutase, the solution containing 6-oxidophamine and desferioxamine is added to the final concentration. 2.5 10 M and 5 M, respectively. The contents of the sample are mixed and at room temperature (18-25 ° C) an increase in the optical density of the sample is recorded at a wavelength of illumination of 490 nm (absorption maximum) over time or after a certain time period. The enzyme activity is judged by the degree of inhibition of the color development, as is customary in other known methods.

Example. The following reagents were prepared: 1-0.1 M phosphate buffer with pH 7.5; 2-5

WITH

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 M solution of 6-oxyfamine on 0.01 M HCI, stabilized with desferrioxamine (1 M), which is prepared immediately prior to the experiment. To do this, we prepared a portion of 6-oxido-aminobromide (2.5 mg) prepared with an accuracy of 0.01 mg, dissolved in 2 ml of a 0.01 M solution of HCl and 1.32 mg of desferoxamine (MM 659) was added. The solution was stirred and sufficient reagent 2 is obtained for 20 samples.

A preparation containing superoxide dismutase (SOD) of human blood is prepared by the method proposed by J.M. McCord and I. Fridovlch.

2.9 ml of 0.005 M Tris-HCf buffer, pH 7.5, are added to 0.1 ml of red blood cell mass. Leave for 0.5 hours in an ice bath to form a hemolysate. 0.4 ml of chloroform-methanol (3: 5) mixture is added to 1 ml of hemolysate, shaken vigorously and centrifuged for 5 minutes at ZOOOd.

The supernatant thus freed of hemoglobin is used as a partially purified blood SOD preparation.

To construct the calibration graph required for calculating the enzyme activity according to the degree of inhibition of autooxidation of 6-oxyfamine, a number of samples are prepared in which different volumes of SOD preparation are added to 1.5 ml of reagent 1. containing different amounts of enzyme. The sample volume is adjusted to 1.9 ml with 0.005 M Tris-HCl buffer, pH 7.5. 0.4 ml of the specified buffer was added to the control sample instead of the preparation containing SOD. The reaction is started by adding 0.1 ml of reagent 2. The change in the optical density of the incubation medium over time is recorded at a light wavelength of 485 nm at room temperature.

Studies have shown that the degree of inhibition of the oxidation of 6-oksidofamina in the presence of desferrioxamine varies in proportion to the concentration of superoxide dismutase, which allows you to build a normal calibration graph of the degree of inhibition of the formation of a colored product in the medium from the amount (concentration) of the enzyme to calculate its activity in conventional units .

The presence of desferrioxamine in the initial solution of 6-oxyfamine protects the latter from auto-oxidation by oxygen dissolved in water, which eliminates the procedure for deaeration of the reagent. At the same time, in the incubation medium of desferioxamin, it inhibits the side reactions of oxidation of 6-oxyfamine, which do not include its interaction with the superoxide radical, which significantly increases the specificity of the method compared to other known methods for determining the activity of the enzyme.

The use of other known chelators of metal ions, such as ethylene diamimmetra tetraacetate (EDTA) and serum albumin, does not protect 6-oxophamine from auto-oxidation, but, on the contrary, accelerates it and somewhat reduces the specificity and sensitivity of the method for determining the activity of superoxide dis- Tazy. The highest activity of superoxide dismutase, which is determined by the proposed method, is shown at pH 7.0-7.5, which allows determination at pH, corresponding to its physiological indicators in the blood and most other tissues of the body. The proposed method carried out the determination of the activity of superoxide dismutase in both purified enzyme preparations and in a partially purified biomaterial (hemoglobin-free hemolysate of human and animal blood, brain hemogenate).

The proposed method is characterized by high sensitivity, accuracy and specificity.

Claims (1)

Claims The method for determining superoxide dismutase activity in biological material by incubating the sample under test in a buffer system containing a superoxide anion radical donor and a chelating agent, followed by photometric measurement of the content of the autooxidation product of the donor under the influence of the superoxide anion radical generated by it, characterized by a superoxide anion radical, which is different from the superoxide anion radical that differs from that of the sample. that, in order to increase the specificity and sensitivity of the method, the incubation is carried out in phosphate buffer at pH 7.0-7.5, and is used as a chelating agent Desferrioxamine is used, and the superoxide anion radical 6 is used as an auto-acid donor, 6 are oxidamines in concentrations 5 respectively. and 2.5 10 4M. %% inhibition 20 injections II Protein 1 g 25 Sk-njfl %% inpooy loo i L OD 0.5% inhibition 90 1.0 0.1 0.5 1.0-1.5 Figz t Protein j 1.5 ICG ml aprobes C Protein 3 -9 ,-one 2 µg.m 11 samples