Abstract
Heparin is shown to bind specifically to the carboxyl terminal region of toxic type I PLA2 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 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 carboxyl 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 the 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.By using Fourier Transformed Infrared (FTIR) spectroscopic measurements, we demonstrate that binding of N-PLA2 to heparin may induce a significant conformational change observed in the amide I region of the enzyme's a-helical and b-sheet structure. It is observed that notable conformational change of N-PLA2 due to heparin binding occurs only when heparin's chain length is at least an octasaccharide as evidenced by Circular Dichroism and optical density measurements.Heparin is shown, for the first time, to be able to promote the interaction between zwitterionic phosphatidylcholine (PC) membrane and N-PLA2, by using fluorescence spectroscopy and Langmuir monolayer binding study. Both the apparent Kd and the binding stoichiometry of lysoPC/N-PLA2 complex change significantly as a result of heparin-induced PC binding. The heparin-enhanced PC binding to N-PLA2 was also observed by the surface area expansion of PC monolayer. Heparin-induced conformational change near Trp60 region is suggested to be mainly responsible for the enhanced binding of N-PLA2 to PC. Since the enzymatic activity of N-PLA2 toward PC/Triton X-100 micelles is not affected, the results suggest a non-enzymatic action mechanism for the toxicity of cobra basic PLA2.