大鼠、小鼠胰蛋白酶的分离纯化及动力学性质

采用STI-Sepharose 4B亲和层析的方法,从鼠新鲜胰脏中分离得到纯的胰蛋白酶。大鼠胰蛋白酶的比活为24 615BAEEU/mg蛋白,总活性回收率47％,小鼠胰蛋白酶的比活为32 768BAEEU/mg蛋白,总活性回收率55％。经SDS-聚丙烯酰胺凝胶电泳鉴定,大鼠、小鼠胰蛋白酶均呈现单一蛋白带,两者的分子量都是24kD。用等电聚焦电泳测定,二者的等电点均为p19.5以上。对它们的动力学性质作了研究,大鼠胰蛋白酶的Km值为2.33×10~(-4)mol/L,K,值为0.92×10~(-5)mol/L,小鼠胰蛋白酶的Km值为5.60×10~(-4)mol/L,K:值为1.27×10~(-5)mol/L。

Separation,Purification and Kinetic Constant of Rat and Mouse Pancreatic Trypsins

Trypsins from rat and mouse fresh pancreas have been isolated and purified by affinity chromatography. The specific activity of trypsin obtained from rat pancreas is 24615 BAEE units per mg protein, and the yield is 47%, and that from mouse pancreas is 32768 BAEE units per mg protein, the yield is 55%. Rat and mouse trypsins prepared by this procedure both appear as a single band upon continous SDS-Polyacrylamide Gel electrophoresis, each with the same molecular weight of 24000 dshons. When these proteins were subjected to isoelectric focusing, both of them focused at pH 9.5. The Km for rat trypsin is 2.33×10~(-4)mol/L (with BAPNA as substrate) and Ki is 0.92 × 10~(-5)mol/L (with banzamidine as inhibitor); for mouse trypsin, Km is 5.60×10~(-4)mol/L and Ki is 1.27×10~(-5)mol/L.