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目的采用凝胶过滤层析法分离全蝎酶解液的抗凝活性部位,并检测活性部分的分子量分布范围。方法采用紫外检测法、考马斯亮蓝法检测,合并凝胶层析蛋白流分;以APTT凝血时间为指标,检验各组分体外抗凝活性;通过MALDI-TOF-MASS法检测分离部分的分子量分布范围。结果全蝎酶解液通过凝胶过滤层析分得的2组分抗凝效果显著,分子量分布范围850~5 300。结论体外抗凝试验表明:凝胶过滤层析法纯化分离全蝎胃蛋白酶酶解液方法可行,MALDI-TOF-MASS表明活性组分为小分子肽类。
Objective To isolate the anticoagulant active site of Scorpion hydrolyzate by gel filtration chromatography and to detect the molecular weight distribution of the active fraction. Methods The UV detection method and Coomassie brilliant blue method were used to detect the protein fractions of gel chromatography. The APTT clotting time was taken as an index to test the anticoagulant activity of each component in vitro. The molecular weight distribution of the fraction was detected by MALDI-TOF-MASS range. Results The scorpion hydrolyzate fraction obtained by gel filtration chromatography showed significant anticoagulant effect and the molecular weight distribution ranged from 850 to 5 300. Conclusion The results of in vitro anticoagulation tests showed that it is feasible to purify pepsin hydrolyzate by gel filtration chromatography. MALDI-TOF-MASS indicates that the active components are small peptides.