土鳖虫胰酶酶解物抗凝活性部位分离纯化及组成分析

Isolation,Purification and Composition Analysis of Anticoagulant Activity Site from Trypsin Hydrolysates of Eupolyphaga sinensis

目的:采用溶剂萃取和凝胶柱色谱法对土鳖虫胰酶酶解物进行分离纯化,初步确定其抗凝活性部位及化学成分组成。方法:应用胰酶酶解法制备土鳖虫胰酶酶解物,以APTT(活化部分凝血酶时间)为指标,采用溶剂萃取法,获得抗凝活性部位IV,经凝胶柱色谱法对其进行分离纯化获得胰酶酶解纯化物;采用考马斯亮蓝法对纯化物进行定量分析,通过MALDI-TOF-MS检测分离部分的分子量分布范围。结果:土鳖虫胰酶酶解物部位IV有较强的抗凝活性,其抗凝活性强度与酶解物质量浓度相关;部位IV经Sephadex G-25纯化后,通过考马斯亮蓝法测得其蛋白质量分数5.5%;进一步经Sephadex G-10脱盐的纯化物,采用MALDI-TOF-MS检测,确定为相对分子质量在850～910 Da的3组多肽混合物。结论:采用凝胶柱色谱法分离纯化土鳖虫胰酶酶解物是可行的,抗凝活性组分是<1 000 Da的小分子肽类。

Objective： To isolate and purify trypsin hydrolysates of Eupolyphaga sinensis by solvent extraction method and sephadex gel column chromatography, and initially identified anticoagulate active components and chemical composition. Method： Rypsin hydrolysates of E. sinensis were prepared by trypsin hydrolysis method, with Active partial thromboplastin time （APTT） as index, anticoagulant active site IV was obtained by solvent extraction method, and purified trypsin hydrolysates were obtained by sephadex G-10 gel column chromatography; Coomassie brilliant blue G-250 was used to quantitative analysis of purified trypsin hydrolysates, and range of molecular weight distribution of separating portion was detected by MALDI-TOF-MS. Result： The composition IV which separated from trypsin hydrolysates of E. sinensis had strong anti-coagulate activity, and the activity was in a positive correlation with the concentration of hydrolysates; After purified by Sephadex G-25, the mass fraction of protein in IV site was 5.5% , which was determined by Coomassie brilliant blue G-250; Purified material was detected by MALDI-TOF-MS after further desalted with Sephadex G-10 gel column, its relative molecular weight range of three groups of polypeptide compounds was identified 850 to 910 Da. Conclusion： It was feasible for separation and purification trypsin hydrolysates of E. sinensis by gel column chromatography, anticoagulant active component was small molecular peptide of less than 1 000 Da.