绵农小麦种子巯基蛋白酶抑制剂的纯化及性质研究

PURIFICATION AND CHARACTERIZATION OF THE CYSTEINE PROTEINASE INHIBITOR FROM MIANNONG WHEAT SEED

绵农11号小麦种子磨粉后的胚和麸皮，经溶液浸取和硫酸铵分级沉淀获得巯基蛋白酶抑制剂（CPI）粗品，再经木瓜蛋白酶－Sepharose4B亲和层析，DEAE－Sepharose离子交换层析和SephadexG－100分子筛层析，可获得两种CPI．通过SDS－PAGE或凝胶过滤法测得其Mr分别为18800［简称CPI（H）］和10500［简称CPI（L）］．它们在PAGE，SDS－PAGE和HPLC上均为单一蛋白带．CPI（L）和CPI（H）对木瓜白酶的抑制活性（ID50）分别为7．8×10－6mol／L和3．40×10－6mol／L．对无花果蛋白酶的抑制活性分别为9．5×10－5mol／L和5．8×10－6mol／L；CPI（H）对菠萝蛋白酶有弱抑制作用，但CPI（L）则无抑制作用；无论是CPI（L）或CPI（H）对胰蛋白酶、胰凝乳蛋白酶和胃蛋白酶均无抑制作用．CPI（H）的N－末端为Ile．CPI（H）和CPI（L）在pH2．0～11．0范围内和90℃处理5min，两种CPI的抑制活性均无变化；CPI（H）和CPI（L）对木爪蛋白酶的抑制曲线经Dixon作图法，得出CPI（H）为反竞争性抑制类型，而CPI（L）为竞争性抑制类型，其Ki值为8．32×10－6mol／L．

Two thiol-protease inhibitors (CPI or cysteine proteinase inhibitors) from seeds of wheat (Miannong) were separeted by extraction, fraction with(NHt)2SO4,affinity chromatography on the papain-Sepharose 4B and ion-exchange chromatography on DEAE-Sepharose and gel filtration on Sephadex G-100 column. Their molecular weights estimated by SDS-PAGE or Sephadex G-100 column were 18 800[CPI H for short]and 10 500[CPI L]. They exhibited one hand on PAGE,SDS-PAGE and HPLC. Both CPI H and CPI L were stable at PH 2. 0-11.0 and below 90℃ for 5 min. The N-terminal amino acid of CPI H was Ile. Both CPI L and CPI H could inhibit papain with ID50 7.8 ×10-6 mol/L and 3. 4 ×10-6 mol/L, respectively. The inhibition of CPI L was competitive with a K. value of 8. 32×10-8 mol/L, but that of CPI H was uncompetitive. CPI H inhibited ficin with ID50 5.8×10-6 mol/L, but gave weak inhibitory effect on bromelin. CPI L also inhibited ficin with ID50 9. 5×10-6 mol/L, but showed no such activity to btomelin. They could not inhibit trypsin,chymotrypsin and pepsin.