Proteolytic specificity of two hemorrhagic factors, LHF-I and LHF-II, isolated from the venom of the bushmaster snake ( Lachesis muta muta)

Abstract

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.