Purification and characterization of a thermostable thrombin-like serine protease from a snake venom

Abstract

Purification and characterization of a thermostable thrombin-like serine protease from a snake venom

Jo, Eu-Ri Advisor : Prof. Lee, Jung Sup Department of Biotechnology Graduate School of Chosun University

Snake venom proteases affect various physiological functions including platelet aggregation, blood coagulation, fibrinolysis, blood pressure, and nervous system. In this study, a thermostable thrombin-like serine protease (TLSP) was purified and characterized from a venom of Gloydius halys ussuriensis (Russian viper). Purification of the TLSP was performed using various chromatographic steps including anion exchange columns with Hiprep 16/10 Q FF and Source 15 Q 4.6/100 PE, a gel filtration with Superdex 75 10/300 GL in order, and then another anion exchange column with Mono QTM HR 5/5 as a final step. The purified TLSP appeared to be a single polypeptide on sodium dodecyl sulfate (SDS)-polyacrylamide gel. The amino terminal sequence of TLSP was found to be NH2-VVGGDEHNIN-COOH, showing a significant similarity ≥ 80% to those of thrombin-like serine proteases from other snake venoms. On SDS-polyacrylamide gel, the apparent molecular weights of TLSP was estimated to be 35 kDa and 26 kDa under reducing and non-reducing conditions, respectively. TLSP was stained with periodic acid-Schiff (PAS) and the size was reduced to about 33 kDa after treatment with N-glycosidase F under the reducing condition. These results suggest that TLSP is a glycosylated protease presumably having N-linked sugar chains. TLSP completely degraded the β-chain of human fibrinogen within 2 hr at 37℃, with much slower digestion of the α-chain. However, TLSP failed to induce formation of fibrin polymer through its fibrinogen cleavage. TLSP cleaved prothrombin, but the resulting products did not show any thrombin activity. In addition, TLSP did not cleave the fibrin polymer. TLSP showed an amidolytic activity against VPR-pNA (specific for thrombin), LGR-pNA (for factor Xa), and S-2288 (for t-PA and a broad serine proteases). Among these substrates, Boc-VPR-pNA was the most favorable one for TLSP. When Boc-VPR-pNA was used as a substrate, the enzyme kinetics of TLSP could be obtained as follows: KM = 0.3 mM and Kcat = 8.69 s-1. The enzyme activity of TLSP was inhibited by various serine protease inhibitors including aprotinin, PMSF, and DFP, not by metalloprotease inhibitors including EDTA, DTT and 1,10-PT. These results suggest that TLSP belongs to a member of the serine proteases. On the other hand, the enzyme activity of TLSP was inhibited by various divalent cations such as Ni2+, Zn2+, and Cu2+. The optimal pH for TLSP enzyme activity was about pH 9.0. Surprisingly, the 59% enzyme activity remained, even at 100℃. Furthermore, TLSP showed no enzyme activity when it was treated with N-glycosidase F, suggesting that a glycosylation on the enzyme may have greatly influence on enzyme activity. Taken together, the results obtained by this study suggest that TLSP is a novel glycosylated thermostable thrombin-like serine protease that cleaves the β-chain more preferentially than the α-chain of fibrinogen.