PROTEOLYTIC SPECIFICITY OF 2 HEMORRHAGIC FACTORS, LHF-I AND LHF-II, ISOLATED FROM THE VENOM OF THE BUSHMASTER SNAKE (LACHESIS-MUTA-MUTA)

Two hemorrhagic metalloproteinases (LHF-I and LHF-II) were previously isolated from Lachesis muta muta (bushmaster snake) venom. The proteol ytic activities of these hemorrhagic factors and of the crude venom we re investigated using as substrate the oxidized B-chain of bovine insu lin. LHF-II cleaves the Ala(14)-Leu(15) bond of insulin B-chain very r apidly and the Phe(24)-Phe(25), His(10)-Leu(11) and His(5)-Leu(6) more slowly, whereas LHF-I hydrolyzed only the Ala(14)-Leu(15) bond. Both hemorrhagic factors cleaved the Leu-Leu bond in the fluorogenic peptid e Abz-Pro-Leu-Gly-Leu-Leu-Gly-Arg-EDDnp. When the insulin B-chain was incubated with crude venom previously treated with 2.5 mM PMSF, the Al a(14)-Leu(15) bond was also rapidly cleaved. In addition, the hemorrha gic activity and the digestion of casein remained unaltered. Both hemo rrhagic and proteolytic activities were inhibited when the crude venom was treated with EDTA, confirming that only metalloproteinases are re sponsible for these activities. The hydrolysis of insulin B-chain and the fluorogenic heptapeptide by these proteinases was found to be in i nverse relationship to their hemorrhagic activities.