MALDI-TOF mass spectrometry analysis of substrate specificity of lebetase,a direct-acting fibrinolytic metalloproteinase from Vipera lebetina snake venom

Lebetase is a direct-acting fibrinolytic zinc metalloendopeptidase related in amino acid sequence to reprolysins which include both hemorrhagic and no n-hemorrhagic proteinases. Despite apparent structural similarities, fibrin olytic and hemorrhagic proteinases differ significantly in substrate specif icity. In this study, we have examined the activity of lebetase I against b iologically active peptides (bradykinin, kallidin, substance P) and 6-10 am ino acid residues containing peptides synthesized according to cleavage reg ions of alpha(2)-macroglobulin, pregnancy zone protein (PZP) and fibrinogen . Lebetase was found to have no activity against studied hexapeptides. Surp risingly, the best substrates for lebetase were substance P, and peptide fr agment of PZP, both were cleaved at position Pro-Gin. Identification of the hydrolysis products of 15 peptides by MALDI-TOF mass spectrometry analysis indicates that lebetase possesses broad substrate specificity. The MALDI-T OF MS technique was proven to be highly efficient for the recovery and iden tification of the peptides released by lebetase hydrolysis. (C) 2000 Elsevi er Science B.V. All rights reserved.