Modification of Carboxylate Groups in Bovine Pancreatic Phospholipase A2

Carboxylase groups in bovine pancreatic phospholipase A2 were modified using a water-soluble carbodiimide and semicarbazide. At pH 5.5 rapid modification of 13 out of the total of 15 carboxylates occurred, leaving Asp-39 and Asp-99 unmodified. Subsequent modification with radioactive semicarbazide at pH 3.5 resulted in the labelling of Asp-39. In the modified proteins both Ca2+ binding and enzymatic activity are completely lost. Modification of the enzyme at pH 5.5 in the presence of Ca2+ ions yielded a protein with three unmodified carboxylates which is capable of binding Ca2+ ions, while 15% residual activity was found. A second reaction, using [14C]semicarbazide at pH 5.5 in the absence of Ca2+, leads to the incorporation of one mole of [14C]semicarbazide/mole of protein with loss of enzymatic activity and Ca2+ binding. The label was found to be attached to Asp-49, demonstrating the essential role of Asp-49 in Ca2+ binding. Studies on the pH dependence of the Ca2+ binding to the protein suggested that Asp-49 has an apparent pK of 5.2.