METHOD OF PRODUCING ANTICOAGULATIVE COLLAGENASE INHIBITOR FROM KING CRAB HEPATOPANCREAS

摘要

FIELD: chemistry; biochemistry.;SUBSTANCE: invention can be used in biotechnology. Material is homogenised in 0.1 M ammonium acetate which contains 0.1-1 mM calcium acetate at pH 6.4-7.0. The homogenate is centrifuged. Ammonium sulphate is added to the supernatant until 60-70% saturation. The obtained complex of enzymes is separated by centrifuging, and then dissolved in distilled water with addition of 0.1-1 mM calcium acetate to obtain a true solution. The solution is then cleaned, concentrated and the enzymes are desalinated on a poly-fibre membrane with pore size of 100 kDa. The supernatant which contains collagenase inhibitor is washed with distilled water, heated to 60-100°C and the precipitated is separated by centrifuging. The obtained overall preparation of collagenase inhibitor is further dissolved in 0.1 M ammonium acetate with pH 6.0-6.4. The precipitate is separated by centrifuging and 35-45% saturated ammonium sulphate is added to the supernatant. The precipitate is separated and dissolved in a 0.01 M tris-buffer with pH 7.0-8.0. The solution is heated to 70-90°C and then centrifuged. The obtained sulphate paste is dissolved in a 0.05 M tris-buffer with pH 8.0 with addition of 0.5 M NaCl. Fractionation is carried out through gel filtration on Sephadex G-100 with subsequent concentration of active fractions through ultrafiltration on a membrane with pore size of 30-50 kDa. The supernatant which contains the desired product is washed and lyophilically dried.;EFFECT: invention widens the range of anticoagulative preparations.;3 cl, 5 dwg, 4 tbl, 1 ex