Purification, characterization and gene cloning of Da-36, a novel serine protease from Deinagkistrodon acutus venom.

Abstract A serine protease termed Da-36 was isolated from crude venom of Deinagkistrodon acutus . The enzyme was a single chain protein with an apparent molecular weight of 36,000 on SDS–PAGE with an isoelectric point of 6.59. Da-36 could clot human plasma by cleaving the A α , B β and γ chains of fibrinogen and also exhibited arginine esterase activity. The proteolytic activity of Da-36 toward TAME was strongly inhibited by PMSF and moderately affected by benzamidine and aprotinin, indicating that it was a serine protease. Meanwhile, Da-36 showed stability with wide temperature (20–50 °C) and pH value ranges (pH 6–10). Divalent metal ions of Ca 2+ , Mg 2+ , and Mn 2+ had no effects but Zn 2+ and Cu 2+ inhibited the arginine esterase activity of Da-36. Total DNA was extracted directly from the lyophilized crude venom and the gene (5.5 kbp) coding for Da-36 had been successfully cloned. Sequence analysis revealed that the Da-36 gene contained five exons and four introns. The mature Da-36 was encoded by four separate exons. The deduced mature amino acid sequence of Da-36 was in good agreement with the determined N-terminal sequence of the purified protein and shared high homology with other serine proteases isolated from different snake venoms. Blast search using amino acid sequence of Da-36 against public database revealed that Da-36 showed a maximal identity of 90% with both Dav-X (Swiss-Prot: Q9I8W9.1 ) and thrombin-like protein 1 (GenBank: AAW56608.1 ) from the same snake species, indicating that Da-36 is a novel serine protease.