Differential binding to phospholipid bilayers modulates membrane-damaging activity of Naja naja atra cardiotoxins

To address the events that modulate membrane-damaging activity of Naja naja atra cardiotoxins (CTXs), the present study was carried out. It was found that CTX isotoxins showed different activities in inducing leakage of vesicles made of egg yolk phosphatidylcholine (EYPC)/dimyristoyl phosphatidic acid (DMPA) or EYPC/egg yolk sphingomyelin (EYSM). Although CTXs had different gross conformations, the toxins showed similar binding affinity for phospholipid vesicles. Topographical contact between toxin molecules and phospholipid vesicles differed for different CTXs as evidenced by fluorescence enhancement of fluorescein-labeled phospholipid. Color transformation of phospholipid/polydiacetylene membrane assay revealed that CTX isotoxins were absorbed on lipid bilayers in different manners. Oxidation of Met residues at the tip of loop II indicated that membrane-bound conformation and orientation of CTXs played a vital role in damaging EYPC/EYSM and EYPC/DMPA vesicles, and suggested that an intact loop II was crucial for inducing leakage of EYSM-containing vesicles rather than that of DMPA-containing vesicles. Moreover, CTXs induced markedly hemolysis of cholesterol-depleted erythrocytes. Taken together, our data indicate that, in addition to membrane organization, membrane-bound conformation and interface-inserted mode of CTXs determine the potency of their membrane-damaging activity.