STUDIES ON PHOSPHOLIPASE A .I. ISOLATION AND CHARACTERIZATION OF 2 ENZYMES FROM CROTALUS ADAMANTEUS VENOM

Two proteins with phospholipase A activity were purified from Crotalus adamanteus venom by a combination of gel filtration; chromatography on a weak cation-exchange resin, DEAE-cellulose, and SESephadex; and crystallization. Both proteins have the same sedimentation coefficient (3.11 S), diffusion coefficient (9.02 × 10-7 cm2/sec), molecular weight (30,000, as determined from sedimentation and diffusion, coefficients and high-speed equilibrium ultracentrifugation), extinction coefficient (E2801%22.7), frictional coefficient (1.16), partial specific volume (0.718 ml/g), and indistinguishable amino acid analyses. However the two proteins are clearly separated on disc gel electrophoresis. Both proteins have specific activities of 3200 μequiv of fatty acid released/min per mg as assayed in ether-methanol solutions using phosphatidylcholine as substrate. There are some unusual features in the amino acid compositions. Out of a total of 266 residues there are 24 residues of glycine, 16 residues of proline, and 15 residues of cystine. There are no detectable free sulfhydryl groups. Both proteins are extremely stable. One enzymatically active protein is not generated from the other during the isolation procedure. All attempts to characterize the difference between the two proteins by tryptic fingerprinting have been unsuccessful. © 1969, American Chemical Society. All rights reserved.