Purification and characterization of fibrolase isoforms from venom of individual southern copperhead (Agkistrodon contortrix Contortrix) snakes.
Fibrolase, a zinc metalloproteinase possessing direct-acting fibrinolytic activity, has been previously purified from southern copperhead (Agkistrodon contortrix contortix) snake venom. We recently reported that a pool of southern copperhead venom from different geographical locations possesses two isoforms of fibrolase (fib1 and fib2) [Loayza, S. L. et al. (1994) J. Chromat. B, in press]. We now report that venom from individual southern copperhead snakes contains the two isoforms which can be separated by a three-step high performance liquid chromatography (HPLC) procedure consisting of hydrophobic interaction chromatography, hydroxylapatite chromatography and weak cation exchange chromatography. Utilizing mass spectrometry we determined that fib1 has a molecular mass of 22,879 atomic mass units (amu) compared to 22,753 amu for fib2. These results support earlier observations during amino acid sequence analysis that a truncated version of the enzyme is produced which is missing the amino-terminal amino acid (< Glu-Arg-Phe-Pro vs. the intact enzyme < Glu-Gln-Arg-Phe-Pro, where < Glu is cyclized glutamine). The truncated version of fibrolase (fib2) has full fibrinolytic activity compared to fib1. EC50 values (concentration of enzyme required to degrade 50% of fibrin in a micro-fibrin plate assay) are 6.4 (+/- 1.0) microM and 5.2 (+/- 0.8) microM for fib 1 and fib2, respectively. Therefore, loss of the amino-terminal amino acid does not appear to influence enzymatic activity. We conclude that the two isoforms of fibrolase arise from variations in the molecular processing of the enzyme by the snake venom gland rather than being caused by the pooling of southern copperhead venoms from different geographical locations.
Trikha
M
M
Department of Biochemistry and Molecular Biology, University of Southern California, School of Medicine, Los Angeles 90033.
Schmitmeier
S
S
Markland
F S
FS
D017836:Agkistrodon; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013058:Mass Spectrometry; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data
C031442:Agkistrodon venoms; D003435:Crotalid Venoms; D007527:Isoenzymes; D005337:Fibrin; D008666:Metalloendopeptidases; C058789:fibrolase