Corvol P, Devaux C, Ito T, Sicard P, Ducloux J, Menard J
Circ Res. 1977 Nov;41(5):616-22. doi: 10.1161/01.res.41.5.616.
Renin was purified from 47 kg of hog kidney to produce enough enzyme for enzymatic and physicochemical characterization. The procedure included extraction at pH 3.5 in the presence of protease inhibitors, two ammonium sulfate precipitations, ion exchange chromatography on Sepharose-hexamethylenediamino-pepstatin gel, gel filtration, and isoelectric focusing. Renin, 2.3 mg, with a specific activity of 1,100 GU/mg of protein was obtained with about 70,000-fold purification and 16% overall recovery. The purity criteria were: (1) a single band on sodium dodecyl sulfate (SDS)-gel electrophoresis, (2) same retardation factor on polyacrylamide gel electrophoresis for renin activity, protein and glycoprotein coloration. Renin was characterized by its stability at--20 degrees C, pH 6.5; its molecular weight on SDS-gel electrophoresis, 36,800; its relative mobility on polyacrylamide gel electrophoresis at pH 7.8; its isoelectric point, 5.15; its amino acid composition, which revealed that renin is a glycoprotein; and its Michaelis constant on tetradecapeptide substrate at pH 6.6, Km = 7.7 X 10(-6) M.
从47千克猪肾中纯化肾素，以获得足够的酶用于酶学和物理化学特性分析。该过程包括在蛋白酶抑制剂存在下于pH 3.5进行提取、两次硫酸铵沉淀、在琼脂糖-六亚甲基二氨基-胃蛋白酶抑制剂凝胶上进行离子交换色谱、凝胶过滤和等电聚焦。获得了2.3毫克肾素，比活性为1100酶活力单位/毫克蛋白质，纯化倍数约为70000倍，总回收率为16%。纯度标准为：（1）在十二烷基硫酸钠（SDS）-凝胶电泳上呈现单一条带；（2）在聚丙烯酰胺凝胶电泳上，肾素活性、蛋白质和糖蛋白染色的迁移率相同。肾素的特性包括：在-20℃、pH 6.5下稳定；在SDS-凝胶电泳上的分子量为36800；在pH 7.8的聚丙烯酰胺凝胶电泳上的相对迁移率；其等电点为5.15；其氨基酸组成表明肾素是一种糖蛋白；以及在pH 6.6下对十四肽底物的米氏常数，Km = 7.7×10⁻⁶ M。
