RU2033169C1 - Process for manufacture of complex including superoxide dis-mutase and catalase from human blood erythrocytes - Google Patents

Abstract

FIELD: medical biochemistry, in particular, enzymology. SUBSTANCE: essence of this method resides in washing out human blood erythrocytes using physiologic salt solution, centrifuging these at 2,500 g during 15 min, lysing erythrocytes with equal amount of distilled water at temperature of -4 C during four hours, adding 0.3 to 0.5 percent by volume of urea to lysate, treating lysate of erythrocytes with admixture of chloroform and ethanol, ratio between lysate, chloroform and ethanol being 1:0.15:0.25, during 40 to 60 min, centrifuging stock, removing precipitate, adding 0.1 normal solution of hydrochloric acid (pH value: 3.7 to 4.2) to supernatant liquid, standing precipitate during 10 to 12 h, repeatedly centrifuging resultant product, adding acetone to thus prepared supernatant liquid (ratio: 1: 1) at temperature of -4 C, repeatedly centrifuging stock, dissolving precipitate in phosphate buffer solution having pH value of 7.4 to 7.8, carrying out dialysis against phosphate buffer, and finally carrying out sterilizing filtration of desired product. End product contains one fraction of catalase and two fractions of superoxide dis-mutase. Electrophoretic purity of end product equals to 99 % and contents of superoxide dis-mutase, catalase and impurities amount to 33.3 %, 66.6 % and 0.1 %, respectively. EFFECT: disclosed method allows reducing time needed for manufacturing end product as compared with prior art (50 to 56 h against 26 to 36 h) and intensifying processing.

Description

 The invention relates to medical biochemistry, in particular to enzymology.

 The aim of the invention is the intensification of the method.

 The method is as follows.

5 l of human blood erythrocytes washed 0.85-1.0% solution of sodium chloride, centrifuged at 2500g for 15 min, lysed erythrocytes with an equal volume of distilled water for 4 hours at ⁴ ° C. The lysate was added ions Cu ^{+ 2} and Zn ⁺² (based on 50 mg of copper acetate and 200 mg of zinc acetate per 1 liter of mixture). To stabilize the catalase enzyme, 0.3-0.5% urea by volume is added to the mixture, stir thoroughly for 15 minutes. Then the lysate of erythrocytes treated with a mixture of chloroform and ethanol, cooled at -20 ^{° C,} in a ratio of lysate-chloroform-ethanol 1: 0.15: 0.25 under stirring for 40-60 min. Centrifuged at 2500g, remove the hemoglobin precipitate. To the supernatant add 0.1 N. a solution of hydrochloric acid to a pH of 3.7-4.2. The precipitate is formed within 10-12 hours, re-centrifuged. To the resulting supernatant containing the desired product were added acetone in a ratio of 1: 1 at a temperature of minus 4 ° ^C. The precipitate maturation time 2 h further centrifuged at 2500g for 20 min.. The precipitate was dissolved in 80-100 ml of KNa-phosphate buffer with a pH of 7.4-7.8. Then dialysis against 0.15 M phosphate buffer followed by sterilizing filtration of the target product through EC plates.

 The target product contains 1 fraction of catalase and 2 fractions of superoxide dismutase. Electrophoretic purity 99% superoxide dismutase content 33.3% catalase 66.6% impurities 0.1% Specific superoxide dismutase activity 2500-3000 units per 1 mg of protein, catalase 30000-40000 units. activity per mg protein.

PRI me R. 5 l of human red blood cells are washed with 0.85% sodium chloride solution, centrifuged at 2500g for 15 minutes. Then lysed erythrocytes with an equal volume of distilled water (5 l) for 4 hours at 4 ° ^C. The lysate was added 500 mg of acetic acid and 2 g of copper acetic acid zinc. Then 50 g of urea is weighed into the lysate, thoroughly stirred for 15 minutes. The mixture (10 l) was added 2.5 liters of alcohol and 1.5 L of chloroform cooled to ^-20 ° C under continuous stirring for 40 min. Then centrifuged at 2500g, remove the precipitate. The supernatant is adjusted to pH 0.1 N. hydrochloric acid solution to pH 4.0. A precipitate is formed for 11 hours. The formed precipitate is removed by centrifugation at 2500g. From the obtained supernatant (7.5 L), a complex of enzymes precipitated with chilled acetone in an amount of 7.5 L. The precipitate matures for 2 hours. Re-centrifuged at 2500g for 20 minutes. The precipitate was dissolved in 100 ml KNa-phosphate buffer. Then dialysis against 0.15 M phosphate buffer followed by sterilizing filtration of the target product.

 The proposed method allows to accelerate the process of obtaining the target product in comparison with the prototype from 50-56 to 26-36 hours

Claims (1)

 METHOD FOR PRODUCING THE SUPEROXIDE DISMUTASE AND CATALASE COMPLEX FROM HUMAN BLOOD RYTHROCYTES by washing the red blood cells with physiological saline, centrifuging for 15 minutes, treating the red blood cell sediment with an equal volume of distilled water, followed by purification of the lysate, which has been sterilized erythrocyte sediment with distilled water, urea is added to the lysate in a final concentration of 0.3 0.5%, incubated for 15 minutes, then the lysate is treated with chloroform orm: ethanol in the ratio lysate: chloroform: ethanol 1 0.15 0.25 with continuous stirring, centrifuged to the supernatant, add 0.1 N hydrochloric acid solution to a pH of 3.7-4.2, incubated for 10-12 hours centrifuged again, to the supernatant containing the target product, add acetone in a ratio of 1 to 1.