Phospholipase A2 (EC 3.1.1.4) was isolated from lyophilized Crotalus atrox venom by chromatography on Sephadex G-75, DEAE-cellulose chromatography and isoelectric focussing in a pH gradient, as a homogeneous protein. In confirmation of an earlier report (Hachimori, Y., Wells, M. A., and Hanahan, D. J. (1971) Biochemistry 10, 4084–4089) the enzyme had a molecular weight of 28 850 and contained two tryptophan residues per mole. At neutral pH the enzyme is a dimer of two identical polypeptide chains which dissociate at low pH. The tryptophan residues in the dimer reside in a restricted, hydrophobic environment, which is disrupted on dissociation into monomers. The circular dichroism (CD) spectrum of the enzyme at neutral pH exhibits a characteristic 1Lb tryptophan spectrum in the aromatic region, with peaks of positive ellipticity at 276, 282 and 290 nm, which are lowered and broadened at low pH, and the difference absorption spectra (pH 7.5 vs pH 2.0) exhibit a prominent peak at 290 nm. No change in the CD spectrum in the peptide region was found at low pH, or in the presence or absence of calcium ion, and a rather low helix content (9%) was estimated from such spectra. The enzyme requires calcium ion for activity, and a dissociation constant for the enzyme–calcium complex of about 10−3 M was estimated from kinetic data. Binding of Ca2+ and the inhibitory cations Hg2+ and Zn2+ was accompanied by perturbation of the tryptophanyl CD spectrum characteristic of exposure of tryptophans to the solvent, but there was no change in the absorption spectrum. The enzyme binds 1-anilinonaphthalene-8-sulfonate (ANS). In the presence of 10−3 M calcium ion, only one ANS binding site was found with Kdiss = 11.9 × 10−6 M, while in the absence of calcium a cooperative binding isotherm was observed, and two molecules of ANS appeared to be bound sequentially with Kdiss values of about 11.7 × 10−6 M and 0.14 × 10−6 M, respectively. The ANS binding site in the presence of 10−3 M calcium ion was more hydrophobic than those in the absence of calcium. At 0.02 M calcium ion in the presence of ANS, a further conformational change in the protein occurred, detected by a large increase in the quantum yield of ANS fluorescence. A structural hypothesis is advanced which qualitatively accounts for these observations.
Recognition of cells in mitosis by flow cytofluormetry.