pH dependence of the binding constant of Ca2+ to the phospholipase A2 of A. halys blomhoffii. Participation of ionizable groups with pK values of 5.16, 6.45, and 7.30.

The pH dependence of the binding constant of Ca2+ to the phospholipase A2 of A. halys blomhoffii was studied at 25 degrees C and an ionic strength of 0.1 by the tryptophyl fluorescence method and aromatic circular dichroism (Ikeda and Samejima (1981) J. Biochem. 89, 1175-1184), and was compared with those for cobra venom phospholipases A2 (Teshima et al. (1981) J. Biochem. 89, 13-20) and for porcine pancreatic enzyme (Pieterson et al. (1974) Biochemistry 13, 1439-1445). The shape of the pH-dependence curve was closer to that for the porcine enzyme than those for the cobra enzymes. The data were analyzed on the basis of our previous findings (Ikeda and Samejima (1981) J. Biochem., 90, 799-804) that the pK value of the ionizable group (alpha-amino group) is perturbed from 7.30 to 6.30 on the Ca2+ binding, and that the protonation of another group corresponding to Asp 49 of the porcine enzyme with a pK value of 5.16 competes with the Ca2+ binding. An additional ionizable group with a pK value of 6.45 was found to participate in the Ca2+ ion binding, and this was assigned to the His residue corresponding to His 48 in the active site of the porcine enzyme.