PURIFICATION AND CHARACTERIZATION OF FIBROLASE ISOFORMS FROM VENOM OFINDIVIDUAL SOUTHERN COPPERHEAD (AGKISTRODON CONTORTRIX CONTORTRIX) SNAKES

Fibrolase, a zinc metalloproteinase possessing direct-acting fibrinoly tic activity, has been previously purified from southern copperhead (A gkistrodon contortrix contortix) snake venom. We recently reported tha t a pool of southern copperhead venom from different geographical loca tions possesses two isoforms of fibrolase (fib1 and fib2) [LOAYZA, S. L. et al. (1994) J. Chromat. B, in press], We now report that venom fr om individual southern copperhead snakes contains the two isoforms whi ch can be separated by a three-step high performance liquid chromatogr aphy (HPLC) procedure consisting of hydrophobic interaction chromatogr aphy, hydroxylapatite chromatography and weak cation exchange chromato graphy. Utilizing mass spectrometry we determined that fib1 has a mole cular mass of 22,879 atomic mass units (amu) compared to 22,753 amu fo r fib2. These results support earlier observations during amino acid s equence analysis that a truncated version of the enzyme is produced wh ich is missing the amino-terminal amino acid (< Glu-Arg-Phe-Pro us. th e intact enzyme < Glu-Gln-Arg-Phe-Pro, where < Glu is cyclized glutami ne). The truncated version of fibrolase (fib2) has full fibrinolytic a ctivity compared to fib1. EC(50) values (concentration of enzyme requi red to degrade 50% of fibrin in a micro-fibrin plate assay) are 6.4 (/-1.0) mu M and 5.2 (+/-0.8) mu M for fib1 and fib2, respectively. The refore, loss of the amino-terminal amino acid does not appear to influ ence enzymatic activity. We conclude that the two isoforms of fibrolas e arise from variations in the molecular processing of the enzyme by t he snake venom gland rather than being caused by the pooling of southe rn copperhead venoms from different geographical locations.