RESOLUTION OF ISOFORMS OF NATURAL AND RECOMBINANT FIBROLASE, THE FIBRINOLYTIC ENZYME FROM AGKISTRODON CONTORTRIX CONTORTRIX SNAKE-VENOM, AND COMPARISON OF THEIR EDTA SENSITIVITIES

Fibrolase, the fibrinolytic enzyme from Agkistrodon contortrix contort rix snake venom, is a zinc metalloproteinase with a molecular mass of 23 kDa. We report a method to isolate two isoforms of natural fibrolas e (fib1 and fib2) and three isoforms of recombinant fibrolase (r-fib1, r-fib2 and r-fib3) using CM 300 cation-exchange high-performance liqu id chromatography. Utilizing mass spectrometry we characterized differ ences in molecular masses of the isoforms of r-fibrolase. These findin gs suggest that the isoforms differ by minor sequence variations at th eir amino-termini. Since the stability of fibrolase is exquisitively s ensitive to the removal of zinc, we examined the EDTA sensitivity of t he isoforms of fibrolase and r-fibrolase to determine if their differe nt chromatographic behavior is related to differences in their zinc af finities. All of the isoforms examined appear to have similar zinc bin ding affinities. Thus, the IC50 (concentration of EDTA to produce 50% inhibition of enzymatic activity) for fib1 is 160 mu M. For the closel y related r-fib1, the IC50 is 180 mu M. Similarly, r-fib3 has an IC50 of 140 mu M.