- 期刊
洋紫荊胰蛋白酶抑制劑對胰蛋白酶抑制作用之動力學研究
Kinetic Studies of the Inhibitory Effects of a Bauhinia purpurea Trypsin Inhibitor on Trypsin
摘要
植物蛋白酶抑制劑是一種天然的抗蟲防禦性蛋白，藉由抑制昆蟲消化道絲胺酸蛋白酶的活性，抑制幼蟲的生長和發育。在腫瘤抑制，抗寄生蟲和抗生素方面，植物蛋白酶抑制劑具有潛在的應用價值。本研究是從豆科植物洋紫荊（Bauhinia purpurea）種子中，利用硫酸銨分割（70-90%）、Sephadex G-50膠體分離管柱、DEAE cellulose陰離子交換樹酯及Trypsin-Sepharose 親和性色層分析法，可純化出洋紫荊胰蛋白酶抑制劑（Bauhinia purpurea trypsin inhibitor, BPTI），以12% SDS-PAGE分析，由單一條多肽鏈組成，分子量約20 kDa，屬於Kunitz-type胰蛋白酶抑制劑；對胰蛋白酶活性的抑制作用莫耳數比為1:1。利用Lineweaver-burk double reciprocal plot及Dixon plots研究其動力學特性，結果顯示BPTI 對胰蛋白酶活性的抑制是屬於競爭性抑制作用，抑制常數（inhibition constant，Ki）為3.82 × 10^(-8) M。
並列摘要
Plant protease inhibitors have been studied to determine their mechanism of action against serine proteinases. Such inhibitors also participate in diverse biological activities, including plant storage, cancer protection, parasite inhibition, and bacterial inhibition. Moreover, they form a critical group of defense proteins in plants because of their ability to inhibit serine proteinase digestive enzymes from insects, thereby suppressing larval growth and development. This study presents a Kunitz-type trypsin inhibitor purified from Bauhinia purpurea seeds. The purification procedure involved by 70%- 90% ammonium sulfate precipitation, Sephadex G-50 column, DEAE ion-exchange column chromatography, and trypsin-Sepharose 4B affinity chromatography. A molecular weight of 20 kDa and a single polypeptide chain were estimated by 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis. Kinetic studies demonstrated that the inhibitory effect of BPTI on trypsin can be categorized as competitive inhibition, with the inhibition constant Ki being 3.82 ×10^(-8) M.