Comparative study of three proteinases from the venom of the Chinese habu snake ( Trimeresurus mucrosquamatus)

Abstract

1. 1. Three immunochemically distinct proteinases (P-1, 2 and 3) devoid of hemorrhagic activity were isolated from the lyophilized venom of Trimeresurus mucrosquamatus using column chromatography on Sephadex G-100, CM-Sephadex C-50, DEAE-Sephacel, CM-Cellulose and Bio-Rex 70. 2. 2. By these procedures, about 7.6, 7.3 and 8.2 mg of purified P-1, 2 and 3 may be obtained from 1 g of crude venom, respectively. 3. 3. The purified proteinases 1–3 were homogeneous by disc electrophoresis on polyacrylamide gel at pH 4.3, isoelectric focusing and by the presence of one precipitin line on immunodiffusion. The isoelectric point of P-1 was 8.1; P-2, 9.2; P-3, 9.8. 4. 4. The molecular weights of proteinases 1–3 were determined to be 23,000, 23,500 and 23,000, by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, respectively. 5. 5. The purified proteinases 1–3 possessed caseinolytic and fibrinogenolytic activities. These activities were inhibited when the proteinases were incubated with the metal chelators ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline or cysteine, but not with egg white trypsin inhibitor (EWTI) or soybean trypsin inhibitor (SBTI). 6. 6. P-1 cleaved the Bβ-chain of fibrinogen first and then the Aα-chain, whereas P-2 and 3 cleaved the Aα-chain first and then the Bβ-chain. However, these three proteinases did not hydrolyze the γ-chain.