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%（NH₄）₂SO₄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 RhizomaPinelliae protein was purified from the 40%(NH) 2SO 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 1.4×1 04, its specific activity was 105 9.01 2 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 Rhizozna Pinelliae protein to trypsin was 1l4.72, and the Ki value, the inhibition constant was about 7.17×10-mol/L． Conclusion: Active Rhizoma Pinelliae protein iS a kind of serine protease inhibitor, a competive inhibitor to trypsin．

山东省教育厅资助项目(J08L17);山东省优秀中青年科学家奖励基金资助项目(2007BSB14087);山东省中药理论与技术创新团队资助项目