Heparin binding to cobra basic phospholipase A2 depends on heparin chain length and amino acid specificity

Heparin is shown to bind specifically to the carboxy-terminal region of toxic type I phospholipase A2 from Naja nigricollis (N-PLA2) by competition assay using synthetic polypeptides and heparin affinity chromatography. The binding strength is seen to depend on heparin chain length and the presence of N-sulfate groups of heparin. It is observed that both electrostatic and non-electrostatic interactions are involved in the specific binding of heparin to the carboxy-terminus. When heparin's size is at least a decasaccharide, about two molecules of N-PLA2 bind to one molecule of heparin, as evidenced by the chemical estimate of protein to carbohydrate ratio in such N-PLA2/heparin complexes. Based on such a stoichiometric measurement and computer modeling of the N-PLA2/heparin complex, it is suggested that the binding sites of the two N-PLA2 molecules on one heparin molecule lie on the opposite sides of the heparin chain.