Ethoxyformylation and guanidination of snake venom phospholipases A2: effects on enzymatic activity, lethality and some pharmacological properties.

Lysine residues in the basic and relatively toxic N. nigricollis phospholipase A2 and in the acidic and relatively nontoxic N. n. atra phospholipase A2 were modified by acylation with ethoxyformic anhydride (in the presence or absence of the substrate dihexanoyl lecithin) or guanidination with O-methylisourea. Ethoxyformylation gave rise to some protein fractions in which enzymatic activity was preserved to a greater degree than intraventricular lethality. Guanidination had little effect on the isoelectric point or catalytic activity of either enzyme or on the lethal potency of the N. n. atra enzyme. However, the intraventricular lethality of the N. nigricollis enzyme was decreased much more than was its intravenous lethality, direct hemolytic potency, anticoagulant activity or cardiotoxic action on rat atrium. These results are compared to those previously obtained when the lysines in these two enzymes were carbamylated with potassium cyanate, a procedure which markedly decreased the isoelectric point of the enzymes. It is concluded that charge alone does not account for differences in toxicity. The data also indicate that there are at least two distinct active sites in both enzymes, one being primarily responsible for enzymatic activity and the other(s) associated with lethal and pharmacological effects of the protein. Modification of lysines affects the latter site(s), while having little or no effect on enzymatic activity.