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目的研究抗凝血肽的制备、分离方法及其体外抗凝血、溶栓效果。方法用木瓜蛋白酶、菠萝蛋白酶、中性蛋白酶和碱性蛋白酶对酪蛋白进行4酶混合水解制备抗凝血肽,以固定化凝血酶对抗凝血肽进行萃取,用新西兰兔进行体外抗凝血、溶栓实验。结果实验确定的抗凝血肽制备条件为酪蛋白质量浓度15%,水解每克酪蛋白的木瓜蛋白酶、菠萝蛋白酶、中性蛋白酶和碱性蛋白酶用量分别为1 500、2 400、1 000、1 250 U,水解温度50℃,pH 7.0,水解时间4 h。固定化凝血酶萃取抗凝血肽的初始浓度6 ATU/mL,萃取温度30℃,pH 5.0,萃取时间30 min。反萃取温度30℃,pH 7.6,反萃取时间40 min。抗凝血肽经高效体积排阻色谱分析,主要成分分子大小与马尿酰-组氨酰-亮氨酸相当。体外抗凝血时间超72 h,溶栓时间24 h。结论酪蛋白抗凝血肽主要成分为3肽,体外抗凝血及溶栓实验效果良好。
Objective To study the preparation and isolation of anticoagulant peptide and its anticoagulant and thrombolytic activity in vitro. Methods Using papain, bromelain, neutral protease and alkaline protease, casein was hydrolyzed by 4 enzymes to prepare anticoagulant peptide. The antithrombotics were extracted by immobilized thrombin. Hitch experiment. Results The experimental conditions for the determination of anticoagulant peptides were as follows: the concentration of casein was 15%, the amount of papain, bromelain, neutral protease and alkaline protease hydrolyzed per gram of casein were 1500, 2400, 250 U, hydrolysis temperature 50 ℃, pH 7.0, hydrolysis time 4 h. The initial concentration of immobilized thrombin extracted anticoagulant peptide 6 ATU / mL, extraction temperature 30 ℃, pH 5.0, extraction time 30 min. Stripping temperature 30 ℃, pH 7.6, anti-extraction time 40 min. High-performance size exclusion chromatography analysis of anticoagulant peptides, the main component of molecular size and hippuramyl-histidyl-leucine quite. Anti-clotting time in vitro over 72 h, thrombolytic time 24 h. Conclusions The main component of casein anticoagulant peptide is 3-peptide. The anti-coagulation and thrombolysis in vitro are effective.