黑眶蟾蜍皮肤白蛋白的纯化及胰蛋白酶抑制活性

目的]探讨黑眶蟾蜍皮肤白蛋白的纯化及胰蛋白酶抑制活性。方法]通过离子交换层析和凝胶过滤层析，从黑眶蟾蜍皮肤中得到其白蛋白，命名为BufomA-skin。65nmol／L的BufomA-skin分别与30nmol／Ltrypsin在25℃不同保温时间（0．25—24h）下测定胰蛋白酶抑制活性，计算剩余酶活力。结果]BufomA-skin为单链蛋白，分子量约为66．7kDa。但在非还原状态下，50～66kDa条带呈弥散样。该蛋白有抑制胰蛋白酶水解小肽底物的活性，但对其他丝氨酸蛋白酶如凝血酶、糜蛋白酶、弹性蛋白酶及枯草杆菌蛋白酶无抑制。BufomA-skin与胰蛋白酶保温24h后，胰蛋白酶活性无恢复。结论]黑眶蟾蜍皮肤白蛋白具有稳定的胰蛋白酶抑制活性，在黑眶蟾蜍防御天敌捕食的过程中发挥重要作用.

Purification of Albumin from Bufo melanostictus Skin and Inhibitory Activity of Its Trypsin

Objective] The aim was to discuss the purification of albumin from Bufo melanostictus skin and trypsin inhibitory activity.Method] On the base of ion-exchange chromatography and gel filtration chromatography,the abumin was obtained from the Bufo melanostictus,which was named as BufomA-skin.The trypsin inhibitory activity was measured on 65 nmol/L BufomA-skin and 30 nmol/L trypsin resp.under 25 ℃ for different temperature preservation time(0.25-24 h) and the residual enzyme activity was calculated.Result] BufomA-skin was single chain protein and its molecular weight was about 66.7 kDa.But under the non reduction state of SDS-PAGE,the 50-66 kDa band showed the dispersive samples.This albumin had the activity of inhibiting trypsin to hydrolyze small peptide substrate,but it had not inhibitive effect on others ammonia pepsinase such as thrombin,chymotrypsin,elastase and subtilisin.Trypsin activity had not recovery after the temperature of BufomA-skin and trypsin were kept for 24 h.Conclusion] The albumin from Bufo melanostictus skin had stable trypsin inhibitory activity,so it could play the important effect during the processing that Bufo melanostictus defended natural enemy predation.