PHOSPHOLIPASE A2 INHIBITORS IN Bothrops neuwiedii diporus VENOM.

DINAPOLI, HERNÁN; DE ROODT, ADOLFO; SEGRE, LILIANA; DOLAB, JORGE; GOULD, EDUARDO; GOULD, JORGE; TROIANO, JUAN CARLOS; AMANTINI, E; VIDAL, JUAN CARLOS

Praga

Congreso; Third World Congress of Herpetology; 1997

Phospholipase A2 (PLA2) activity was measured in crude B. Neuwiedii diporus venom on 10.0 ml of egg yolk lipoprotein (25 mM lipid P) 0.2 M NaCI, 10 mM CaCI2 at 22°C using a pH-Stat (Radiometer). The initial rate increases as an hyperbola with the venom concentration and consequently, the PLA 2 specific activity decreases as the venom concentration increases. This behavior is not due to substrate depletion; it depends on dilution and is not affected by substrate concentration. Kinetic analysis suggests that it can be explained by the presence of an endogenous, non competitive PLA2 inhibitor of the "tight binding" type normally present in the venom at a molar concentration close to that of the enzyme with a Ki = 8.19 (± 1.8) x1O -s M . The enzyme, which constitutes about 8% of the venom protein can be purified and separated from the inhibitor in a two .step procedure consisting of (1) Gel-filtration on Sephadex G-50 in 0.1 M ammonium formate .buffer pH 4.5 and (2) Gel-filtration on Sephadex G-25 in 10 mM phosphate-Tris pH 7.6, whcre the enzyme is eluted in the void volume. After this step, measurement of PLA2 activity shows that the initial rate is a linear function of the enzyme concentration: the PLA2 specific activity is constant and independent of the enzyme concentration; the addition of the fractions eluted in the included volume results in inhibition of the PLA2 activity and the total activity units recovered is 980% of the total units in the crude venom sample.