The soluble wild type and recombinant proteins expressed in tobacco seeds, were extracted as previously described (Grossi-de-Sá et al., 1997) by grinding 150 mg of dry seeds, in an ice-cold mortar with 1.0 mL 50 mM Tris-HCl, pH 8.0, containing 0.1% Triton X-100, 30 mM NaCl and 1% 2-mercaptoethanol. The homogenates were twice centrifuged for 10 minutes at the top speed of a bench centrifuge and the supernatants stored at -20ºC or immediately used to measure the inhibitory activity. Protein concentration was determined according to Bradford (1976) using bovine serum albumin as standard