Purification and activation of phospholipase A2 isoforms from Naja mossambica mossambica (spitting cobra) venom

Isoforms of phospholipase A2 (PLA2) from the venom of Naja mossambica mossambica (the spitting cobra) were purified by a combination of gel filtration on Bio-gel P-30 and ion exchange chromatography on DE-52 Cellulose and the purification followed by three types of polyacrylamide gel electrophoresis. SDS PAGE failed to resolve the active band into separate isoforms. Acid/urea PAGE, resolved the peptides and major protein components of the venom and was able to separate two PLA2 bands in the whole venom. Alkali/urea PAGE resolved four PLA2 bands in whole venom, but could resolve six distinct purified PLA2 species. Of the known isoforms, the acidic form (CM-1) was purified to homogeneity. The basic non-toxic isoform (CM-II) was shown to migrate as a close doublet of PLA2 isoforms. A novel minor purified isoform was identified with mobility intermediate between CM-I and the basic non-toxic isoform CM-II. CM-III was shown to contain a minor PLA2 contaminant. The analysis was facilitated by the fact that all of the isoforms could be eluted from the gels with > 60% recovery of activity. The venom therefore contains at least six isoforms of PLA2 which differ largely by their content of acidic acids. Oleoyl imidazolide treatment increased the haemolytic activity of all but the toxic PLA2 isoform in Naja mossambica mossambica, but partially inhibited the catalytic activity of acidic, toxic and newly purified isoforms whilst partially activating the non-toxic isoform.