Abstract
The hydrolytic activity of secretory phospholipase A 2 (PLA 2 ) is regulated by many factors, including the physical state of substrate aggregates and the chemical nature of phospholipid molecules. In order to achieve strong binding of PLA 2 on its substrates, many previous works have used anionic lipid dispersion to characterize the orientation and penetration depth of PLA 2 molecules on membrane surfaces. In this study, we applied monolayer technique with controllable surface area to investigate the PLA 2 s of Taiwan cobra venom and bee venom on zwitterionic phophatidylcholine monolayers and demonstrated an optimum hydrolytic activity at a surface pressure of 18 and 24 mN/m, respectively. By combining polarized attenuated total reflection Fourier-transform infrared spectroscopy and monolayer-binding experiments, we found that the amount of membrane-bound PLA 2 decreased markedly as the surface pressure of the monolayer was increased. Interestingly, the insertion area of the PLA 2 s decreased to near zero as the surface pressure increased to the optimum pressure for hydrolytic activity. On the basis of the measured infrared dichroic ratio, the orientation of the PLA 2 s bound to zwitterionic membranes was similar to that observed on a negatively charged membrane and was independent of the surface pressure. Our findings suggest that both PLA 2 s were located on the membrane surface rather than penetrating the membrane bilayer and that the deeply inserted mode is not a favorable condition for the hydrolysis of phospholipids in zwitterionic phospholipid membranes. The results are discussed in terms of the easy access of catalytic water for the PLA 2 activity and the mobilization of its substrate and product to facilitate the catalytic process. © 2011 American Chemical Society.