INFLUENCE OF CHEMISTRY IN IMMOBILIZATION OF COBRA VENOM PHOSPHOLIPASE-A(2) - IMPLICATIONS AS TO MECHANISM

Phospholipase A2 from Naja naja kaouthia venom was covalently coupled onto agarose beads using two different chemistries. The effect of mice llar competitive inhibitors in the coupling media was evaluated. Enzym e bound to N-hydroxysuccinimide-activated agarose, which is reactive p rimarily toward epsilon-amino groups, had 20% activity retention again st micellar diheptanoylphosphatidylcholine (DiC7-PC). Enzyme bound thr ough carboxylic groups, using a modification of the carbodiimide metho d, had 50% retention. Similar relative activities were observed, for b oth conjugates, in monomeric dihexanoyl-PC and in mixed micelles of Tr iton X-100 with dipalmitoyl-PC or dioleoylphosphatidylethanolamine. Th e soluble form of the enzyme showed premicellar activation against mon omeric DiC7-PC, while the immobilized form showed interfacial recognit ion at concentrations around the critical micellar concentration. Thes e results suggest that the enzyme activity lost upon immobilization is a result of the inherent chemical modification of the enzyme and that enzyme oligomerization and interfacial recognition are not cause-effe ct phenomena.