Modification of Arginine Residues in Porcine Pancreatic Phospholipase A 2

Although phenylglyoxal monohydrate reacts with Arg‐6 in porcine pancreatic phospholipase A 2 , concomittantly the α‐amino group of the N‐terminal Ala‐1 residue is quantitatively transaminated. Due to this latter reaction the enzymatic activity toward micellar substrate is lost irrespective of the Arg‐6 modification. Upon reaction of [7‐ 14 C]phenylglyoxal monohydrate with α‐amino‐blocked phospholipase A 2 analogs, two molecules of the reagent were incorporated per protein molecule, which were found to be present on Arg‐6. Removal of α‐amino‐blocking groups after tile modification reaction furnished the corresponding Arg‐6‐modified phospholipases possessing 30–38% of their original specific enzymatic activities in the egg‐yolk assay. After reaction of 1,2‐[1‐ 14 C]cyclohexanedione with porcine phospholipase A 2 the crude reaction mixture was purified by chromatography on quaternary diethyl‐(2‐hydroxypropyl)aminoethyl‐Sephadex in the presence of borate. A fraction was obtained containing a pure protein in which one molecule of 14 C‐labeled reagent per protein molecule was incorporated which was found to be localized almost exclusively on Arg‐6. Cyclohexanedione modification of Arg‐6 in phospholipase A 2 does not significantly influence its catalytic activity when assayed toward monomeric and micellar substrates. The results of direct binding experiments using substrate analogs and of monolayer studies of the phospholipase modified at Arg‐6 by cyclohexanedione are in agreement with previous findings that Arg‐6 is involved in the interaction of the enzyme with lipid‐water interfaces.