Blackburn P, Wilson G, Moore S
J Biol Chem. 1977 Aug 25;252(16):5904-10.
A soluble ribonuclease inhibitor from the human placenta has been purified 4000-fold by a combination of ion exchange and affinity chromatography. The inhibitor has been isolated in 45% yield (about 2 mg/placenta) as a protein that is homogeneous by sodium dodecyl sulfate-gel electrophoresis. In common with the inhibitors of pancreatic ribonuclease from other tissues that have been studied earlier, the placental inhibitor is an acidic protein of molecular weight near 50,000; it forms a 1:1 complex with bovine pancreatic RNase A and is a noncompetitive inhibitor of the pancreatic enzyme, with a Ki of 3 X 10(-10) M. The amino acid composition of the protein has been determined. The protein contains 30 half-cystine plus cysteine residues determined as cysteic acid after performic acid oxidation. At pH 8.6 the nondenatured protein alkylated with iodoacetic acid in the presence of free thiol has 8 free sulfhydryl groups. The inhibitor is irreversibly inactivated by sulfhydryl reagents and also by removal of free thiol from solutions of the protein. Inactivation by sulfhydryl reagents causes the dissociation of the RNase - inhibitor complex into active RNase and inactive inhibitor.
通过离子交换和亲和层析相结合的方法，从人胎盘中纯化出一种可溶性核糖核酸酶抑制剂，纯化倍数达4000倍。该抑制剂的分离产率为45%（约2毫克/胎盘），通过十二烷基硫酸钠 - 凝胶电泳检测为单一蛋白质。与之前研究过的其他组织来源的胰腺核糖核酸酶抑制剂一样，胎盘抑制剂是一种分子量接近50,000的酸性蛋白质；它与牛胰腺核糖核酸酶A形成1:1复合物，是该胰腺酶的非竞争性抑制剂，抑制常数Ki为3×10(-10)M。已测定该蛋白质的氨基酸组成。该蛋白质含有30个半胱氨酸加半胱氨酸残基，在过甲酸氧化后测定为半胱氨酸。在pH 8.6时，在游离巯基存在下用碘乙酸烷基化的未变性蛋白质有8个游离巯基。该抑制剂可被巯基试剂不可逆地灭活，也可通过从蛋白质溶液中去除游离巯基而灭活。巯基试剂导致核糖核酸酶 - 抑制剂复合物解离成活性核糖核酸酶和无活性抑制剂。
