台灣欒樹種子中胰蛋白酶抑制劑之純化及特性分析

Purification and Characterization of a Trypsin Inhibitor from Koelreuteria formosana Seeds

洪志宏(Chih-Hung Hung) ； 董陳峰(Chen-Feng Dong) ； 王海龍(Hai-Lung Wang)

《健康管理學刊》 13卷2期 (2015/12) Pp. 1-12

摘要

由台灣欒樹(Koelreuteria formosana)種子純化出胰蛋白酶抑制劑(Koelreuteria formosana trypsin inhibitor)，簡稱KFTI，純化過程是利用硫酸銨分割(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，以15% SDS-PAGE分析其純度及分子量，得知其分子量約19 kDa，由12.3 kDa 及6.7 kDa 兩條多胜鏈以雙硫鍵連接所組成，屬於Kunitz type 蛋白酶抑制劑。KFTI 對胰蛋白酶抑制作用其莫爾數比為1:1，動力學的分析其抑制胰蛋白酶的活性作用是屬於競爭性的抑制作用，解離常數(dissociation constant, Ki)為1.78 × 10-9 M。進一步對此蛋白的性質研究：以100℃高溫處理10 分鐘，仍具有70%抑制胰蛋白酶的活性，可知KFTI 對熱具有非常高的穩定性；在pH 值3.0-10.0 範圍內反應60 分鐘，顯示KFTI 抑制胰蛋白酶的活性在廣泛的的pH 值環境下，仍保持其結構的穩定性，活性不受pH 值的影響；但在0.2% SDS 作用10 分鐘，尚有不到10％的活性，可見其構造對SDS 非常不穩定; 以5 mM DTT 處理10 分鐘後，KFTI 抑制胰蛋白酶的活性只剩下5%，可知雙硫鍵對其構形的穩定有絕對的重要性。

關鍵字

台灣欒樹胰蛋白酶抑制劑 ； Kunitz-type 蛋白酶抑制劑 ； 解離常數

並列摘要

A trypsin inhibitor (KFTI) was purified from the seed of Koelreuteria formosana by ammonium sulfate precipitation (70-90%), Sephadex G-50 gel filitration, DEAE-cellulose ion-exchange column and trypsin-Sepharose 4B affinity chromatography. 15% SDS-PAGE analysis of KFTI revealed that it is constituted by two polypeptide chains (A-chain, Mr 12.3 kDa and B-chain, Mr 6.7 kDa), the molecular weight being ~19 kDa. The KFTI was found to be a thermostable Kunitz type protein inhibitors. KFTI inhibits bovine trypsin activity in 1:1 molar ratio. Kinetics studies showed that the protein is a competitive inhibitor with a dissociation constant (Ki) of 1.78 × 10^(-9)M. The stability of KFTI was studied by exposing it to altered conditions of temperature, pH, protein-denaturing agents like SDS and reducing agent DTT, and measuring the residual inhibitor activity. The inhibitory activity retained unchanged over a wide range of temeratures (0-90℃) and pH (3-10). KFTI only retained 10% activity after treatment with 0.2% SDS for 10 min and 5% activity after treatment with 5 mM DTT. The stability of KFTI is apparently related to the presence of disulfide bridge.

並列關鍵字

Koelreuteria formosana trypsin inhibitor ； Kunitz-type protein inhibitor ； dissociation constant