Purification and molecular cloning of salmobin, a thrombin-like enzyme from korean salmosa snake (Agkistrodon halys)

Salrnobin, a thrombin-like proteinase has been identified and purified to homogeneity from the venom of Agkistrodon halys (Korean Salmosa snake). It is a single chain glycoprotein with an apparent molecular weight of 43,000 on SDSPAGE and 38,320 by MALDI-mass spectrometry, and an isoelectric point of pH7.6. Salmobin acted on fibrinogen to form fibrin with a specific acyivity of 267 NIH units/mg. It predominantly cleaved the A? chain of fibrinogen. The activity of salmobin was readily inhibited by diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, antipain and 2-mercaptoethanol, indicating it is a serine protease and disulfide bonds will be critical for maintaining its activity. However, the enzyme was not inhibited by aprotinin, hirudin, soybean trypsin inhibitor, a2-antiplasmin and antithrombin III. cDNA library was prepared from the venom glands of A. halys and was screened with oligonucleotide probes designed on (he basis of the N-terminal and the internal peptide sequences of salmobin. The deduced complete amino acid sequence of salmobin indicates that the mature salmobin protein is synthesized as a pre-zymogen of 260 amino acids, including a putative secretory signal peptide of 18 amino acids and a proposed zymogen peptide of 6 amino acid residues. The cDNA sequence indicates that the mature salmobin protein is composed of 236 amino acids and contains two potential N-glycosylation site, Asn-X-Ser/Thr at Asn⁹⁰ and Asn¹³². The sequence of salmobin exhibits a high degree of sequence identity with other snake venom proteases: 78% with gyroxin, 75% with calobin, 67% with ancrod and flaboxobin, 65% with batroxobin, and 62% with RVV-V. Like other thrombin-like enzymes, salmobin contains 12 cysteine residues and it could be predicted that they are all involved in the formation of six disulfide bonds. The homology of salmobin with venom thrombin-like enzymes indicated that the amino acid residues which form the catalytic triad are His43, Asp⁸⁸, and Ser¹⁸².