一种具有胰蛋白酶抑制活性的半夏蛋白的纯化与N端氨基酸序列分析

Purification of a protein with trypsin-inhibiting activity from Rhizoma Pinelliae and analysis of its N-terminal amino acid sequence

目的分离纯化具有胰蛋白酶抑制活性的半夏蛋白,分析其对胰蛋白酶的抑制活性、抑制机制与N端氨基酸序列。方法采用以胰蛋白酶为配体的亲和色谱和Sephadex G-50凝胶过滤法,从半夏蛋白粗提液的40%(NH_4)_2SO_4沉淀中分离纯化活性半夏蛋白;采用12%SDS-PAGE鉴定其纯度,并估算其相对分子质量;采用Ed-man降解法分析其N-端氨基酸序列。结果纯化的活性半夏蛋白经SDS-PAGE检测呈现单一条带,相对分子质量为1.4×10～4;比活力为1059.012U/mg,活力回收率为24.40%;对胰蛋白酶的质量抑制比为1:4.72,抑制常数(Ki)为7.17×10～(-6)mol/L;其N端前6个氨基酸残基顺序为DPVVDG。结论该活性半夏蛋白是一种丝氨酸蛋白酶抑制刺,为胰蛋白酶的竞争性抑制剂。

Objective To purify a Rhizoma Pinelliae protein, determine its inhibiting activity and mechanism to trypsin, and analyze its N-terminal amino acid sequence. Methods Active Rhizoma Pinelliae protein was purified from the 40% （NH4）2SO4 sediment of the crude extracted protein from Rhizoma Pinelliae by affinity chromatography of trypsin-Sepharose CL 4B and gel filtration of Sephadex G-50; 12% of SDS-PAGE was used for determining the purity of the purified Rhizoma Pinelliae protein and estimating its molecular weight. N-terminal amino acid sequence of active Rhizoma Pinelliae protein was analyzed by Edman degradation. Results The purified active Rhizoma Pinelliae protein showed a single band on SDS-PAGE gel ,which estimated molecular weight was about 1.4× 10^4, its specific activity was 1 059. 012 U/mg, and the yield was about 24.40%. The N-terminal sequence of the preceding six amino acid residues of active Rhizoma Pinelliae protein was DPVVDG; The quality inhibiting ratio of active Rhizoma Pinelliae protein to trypsin was 1 ： 4.72, and the Ki value , the inhibition constant was about 7.17×10^-6 mol/L. Conclusion Active Rhizoma Pinelliae protein is a kind of serine protease inhibitor, a competive inhibitor to trypsin.