Characterization of three edema-inducing phospholipase A2 enzymes from habu (Trimeresurus flavoviridis) venom and their interaction with the alkaloid aristolochic acid

A basic phospholipase A2 (PLA2) enzyme, TFV PL-X (pI 9.2) and two acidic PLA2 enzymes, TFV PL-Ia (pI 4.9) and TFV PL-Ib (pI 4.5) were purified from Trimeresurus flavoviridis venom on CM-Sephadex C-25 and QAE-Sephadex A-25 columns, respectively. The basic enzyme exists as a monomer, whereas the acidic enzymes are dimers. These enzymes differ in properties such as molecular weight, Km, optimum pH and temperature and pharmacological properties. The basic enzyme hydrolysed purified phospholipids in the order of PC greater than PE greater than PS greater than PI = 0, while for TFV PL-Ia and TFV PL-Ib the order was PC greater than PE greater than PS = PI = 0. TFV PL-X was comparatively more toxic, with an LD50 value of 4.2 micrograms/g (i.p.), while the acidic PLA2 enzymes had LD50 values above 8 micrograms/g (i.p.). All three enzymes induced edema when injected into the mouse foot pad. Aristolochic acid, an alkaloid (8-methoxy-6-nitrophenanthro(3,4-d)-1,3-dioxole-5-carboxylic acid) from the medicinal plant Aristolochia radix, interacts with these PLA2 enzymes. It is a competitive inhibitor with varying affinity when PC is used as substrate. Aristolochic acid inhibits direct and indirect hemolytic activity, as well as edema-inducing activity, of TFV PL-X, but fails to neutralize the lethal potency of the enzyme. On the other hand, it inhibits direct and indirect lytic activity of TFV PL-Ia and TFV PL-Ib only at 10-fold higher concentrations and it enhances the edema-inducing activity of these enzymes. Such effects of aristolochic acid indicates that (1) different mechanisms may be involved in the edema-inducing activity of PLA2 enzymes and (2) catalytic and pharmacological sites are separate on the PLA2 molecule.