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 contortrix snake venom, is a zinc metalloproteinase with a molecular mass of 23 kDa. We report a method to isolate two isoforms of natural fibrolase (fib1 and fib2) and three isoforms of recombinant fibrolase (r-fib1, r-fib2 and r-fib3) using CM 300 cation-exchange high-performance liquid chromatography. Utilizing mass spectrometry we characterized differences in molecular masses of the isoforms of r-fibrolase. These findings suggest that the isoforms differ by minor sequence variations at their amino-termini. Since the stability of fibrolase is exquisitely sensitive to the removal of zinc, we examined the EDTA sensitivity of the isoforms of fibrolase and r-fibrolase to determine if their different chromatographic behavior is related to differences in their zinc affinities. All of the isoforms examined appear to have similar zinc binding affinities. Thus, the IC50 (concentration of EDTA to produce 50% inhibition of enzymatic activity) for fib1 is 160 microM. For the closely related r-fib1, the IC50 is 180 microM. Similarly, r-fib3 has an IC50 of 140 microM.
Loayza
S L
SL
Department of Biochemistry, University of Southern California, School of Medicine, Los Angeles 90033.
Trikha
M
M
Markland
F S
FS
Riquelme
P
P
Kuo
J
J
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003435:Crotalid Venoms / Q000737:chemistry / Q000201:enzymology*; D004492:Edetic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification*; D013058:Mass Spectrometry; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D015032:Zinc / Q000378:metabolism
C031442:Agkistrodon venoms; D003435:Crotalid Venoms; D007527:Isoenzymes; D011994:Recombinant Proteins; D004492:Edetic Acid; D008666:Metalloendopeptidases; C058789:fibrolase; D015032:Zinc