台灣龜殼花毒液中纖維蛋白原分解酵素之研究

Abstract

經陰離子交換、凝膠過濾及高壓液相層析法，我們由龜殼花粗毒中分離純化出三個纖維蛋白原分解酵素，分別命名為TM-1,TM-2及TM-3。純化之酵素溶液，由聚凝膠電泳及高壓液相層析判斷，都具高純度且分子量在27,000左右。這三個酵素對纖維蛋白原之α-、β-次單元具水解活性，且以α-次單元之水解為優先。酵素活性受pH值影響不大，但會在65℃以上之高溫失去水解能力。由EDTA ,1,10-Phenanthroline能抑制他們活性的現象推斷，這些酵素應都屬於金屬蛋白分解酵素。對胰島素B-鏈為受質之催化專一性，TM-1，-2以Ala(14)-Leu(15）及Tyr(16)-Leu(17）優先水解，而TM-3除此外，尚有His(10)-Leu(11)。當反應時間延長，TM-3不論水解位置、順序乃至於水解活性與TM-1，-2明顯區別。向酵素自體溶解後之產物觀察，TM-1與TM-2之間可能擁有相當類似的一級結構。此外，這三個酵素之纖維蛋白原分解活性、抑制劑影響類型及氨基酸組成與分子量約小4,000左右的Protease-2，-3及α-Fibrinogenase性質相近。

Three fibrinogenolytic enzymes named as TM-1, TM-2 and TM-3 were purified from the venom of Taiwan habu, by using anion-exchange and gel-filtration chromatography followed by cation-exchange HPLC. Purified fractions of TM-1, -2 and -3, as judged by SDS-polyacrylamide gel electrophoresis and HPLC, were shown to be homogeneous with molecular masses of about 27kDa. These enzymes cleaved the α-chain of fibrinogen first and then the β-chain with low activity on the γ-chain. Fibrinogenolytic activities of these enzymes were not significantly affected at extreme pH values when incubated with fibrinogen, activity loss occurred at temperatures higher than 65°C. Enzyme activities were inhibited when incubated with some metal chelators such as EDTA or 1,10- phenanthroline, suggesting that these fibrinogenases belong to metalloproteases. TM-1 and TM-2 cleaved the bonds of Ala(14)-Leu(15) and Tyr(16)-Leu(17) of oxidized insulin B-chain most rapidly while TM-3 also hydrolyzed the His(10)-Leu(11)bond in addition to those of TM-1 and TM-2. Upon longer incubation time of these enzymes with oxidized insulin B-chain, TM-3 was distinguishably less active than TM-1 and TM-2 with regard to cleavage sites and activities of hydrolysis. The identical products with similar molecular weights observed on the autolysis of TM-1 and TM-2 suggest that these two proteases are similar in primary structures. Chemical properties including fibrinogenolytic activities, inhibitor effects and amino acid compositions of these enzymes were similar to those reported for Proteinase-2, -3 and α-Fibrinogenase, the fibrinogenases previously identified from the venom of Trimeresurus mucrosquamatus with molecular weights about 22,500-23,500.