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 proteolytic activities of these hemorrhagic factors and of the crude venom were investigated using as substrate the oxidized B-chain of bovine insulin. LHF-II cleaves the Ala(14)-Leu(15) bond of insulin B-chain very rapidly 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 peptide 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 Ala(14)-Leu(15) bond was also rapidly cleaved. In addition, the hemorrhagic activity and the digestion of casein remained unaltered. Both hemorrhagic and proteolytic activities were inhibited when the crude venom was treated with EDTA, confirming that only metalloproteinases are responsible for these activities. The hydrolysis of insulin B-chain and the fluorogenic heptapeptide by these proteinases was found to be in inverse relationship to their hemorrhagic activities.