PHOSPHOLIPASE A2 FROM CROTALUS ATROX VENOM .I. PURIFICATION AND SOME PROPERTIES

Phospholipase A2 (phosphatide acylhydrolase, EC 3.1.1.4) has been purified from Crotalus atrox (Western diamondback rattlesnake) venom using ammonium sulfate fractionation followed by Sephadex G-75 column chromatography. The active protein, appearing as one band on polyacrylamide gel disc electrophoresis at different pH values (4.5-8.4), is eluted from Sephadex G-75 with a constant specific activity 35 times that of the crude venom, and has no detectable protease, phosphodiesterase, or monoesterase activity. Using rat liver lecithin of known composition of fatty acids at the 1 and 2 positions, the positional specificity of the purified enzyme has been established by the exclusive release of 2-position fatty acids, which are largely unsaturated. The enzyme acts on ovolecithin in ethereal or chloroform solutions or in ultrasonicated aqueous dispersions with complete hydrolysis to lysolecithin plus fatty acids. Michaelis- Menten kinetics are apparently applicable in the ether and chloroform systems, the Km values being 8.3 and 8.5 mM, respectively. The molecular weight, determined by Sephadex G-75 chromatography, appears to be in the range 14,500 ± 500. In contrast, the enzyme from the venom of the closely related snake Crotalus adamanteus has molecular weight about 30,000 (Saito, K., and Hanahan, D. J. (1962), Biochemistry 1, 521). Moreover, only one isozymic form appears to exist in C. atrox venom, while two isozymes are found in C. adamanteus. In ultrasonicated substrate dispersions, the enzyme has a broad pH optimum between 6.8 and 7.6 and a sharp temperature optimum at 46°. Against common belief that calcium ions alone can activate the enzyme, the purified phospholipase A2 is activated to varying degrees by Ca2+, Ni2+, Co2+, Mg2+, and Cd2+ but not by Hg2+, Zn2+, Cu2+, or Ba2+ ions. At fixed Ca2+ ion concentration, increasing concentrations of Zn2+, Cu2+, and Ba2+ produce progressive inhibition, suggesting possible competition between these ions and calcium. The optimal calcium concentration for activity is 0.02 m. The protective role of EDTA in venom solutions has been shown to be attributable to chelation of trace amounts of inhibitory divalent cations. Unlike phospholipase A2 from other sources, the C. atrox enzyme is completely inactivated by diisopropylphosphoryl fluoride and iodoacetate. O-Methyliosourea also inactivates the enzyme; however p-mercuribenzoate and mercaptoethanol produce little inhibition. The purified phospholipase A2 withstands heating to 80° for 30 min at pH 3.0, but similar treatment at pH 7.4 immediately inactivates it. Lyophilization also destroys the enzyme activity, which is slowly regained upon dissolution in aqueous buffer, after a lag phase of several hours. © 1969, American Chemical Society. All rights reserved.