In a previous experiment it was noticed a significant increase of the hemagglutination activity upon leaf injury (data will be published elsewhere). After this, the leaves were powdered in the presence of liquid nitrogen and stored at −80 °C until required. DEAE-cellulose column was obtained from Whatman International

Ltd., Maidstone, England; Phenyl-Sepharose 6-Fast Flow column was obtained from GE Healthcare, Uppsala, Sweden. Morphine was purchased from Sigma Aldrich Chemical (Saint Louis, MO, USA). www.selleckchem.com/screening/protease-inhibitor-library.html The other chemicals were all of analytical grade and obtained from local suppliers. The soluble proteins were extracted from the leaf powder with three volumes of 25 mM Tris–HCl, pH 7.5, supplemented with 3% (w/v) polyvinylpolypyrrolidone (PVPP) and 5 mM ascorbic acid, for 2 h at 4 °C, under gentle shaking. After filtration through nylon cloth, the filtrate was centrifuged at 10,000 × g for 30 min, at 4 °C, and the supernatant (crude extract) recovered. The crude extract was precipitated with ammonium sulfate at 30% saturation (176 g/L) and the suspension maintained at 4 °C for 12 h. The precipitate obtained (Fraction 0–30%, shortly F030) after centrifugation (10,000 × g, Selumetinib 40 min, 4 °C) was dialyzed exhaustively against Milli-Q grade water, lyophilized, and suspended in 25 mM Tris–HCl, pH 7.5. After centrifugation (10,000 × g, 20 min, 4 °C), the fraction

F030 was submitted to ion-exchange chromatography on a DEAE-cellulose column equilibrated with 25 mM Tris–HCl, pH 7.5. The through fraction was eluted from the column with the equilibrating buffer. The retained material was eluted with 25 mM Tris–HCl, pH 7.5, containing 200 mM NaCl, at a flow rate of 1 mL/min, dialyzed exhaustively against water and lyophilized. Next, it was suspended in 25 mM why Tris–HCl, pH 7.5, containing 420 mM of ammonium sulfate, centrifuged (10,000 × g, 20 min, 4 °C), and the supernatant obtained chromatographed on a Phenyl-Sepharose 6-Fast Flow column, equilibrated with the above buffer.

The protein fraction obtained after elution with 25 mM Tris-HCl, pH 7.5, containing 100 mM of ammonium sulfate, at a flow rate of 1 mL/min, was dialyzed against Milli-Q grade water and lyophilized. This material represented the lectin-enriched fraction (LEF) that was characterized and used to assess toxicity. It was determined as previously described (Bradford, 1976). Absorbance at 280 nm was also used to monitor protein elution profiles during chromatographies. Protein fractions were analyzed by polyacrylamide gel electrophoresis (15% running gel, 3.5% stacking gel) (Laemmli, 1970). The samples were solubilized in 125 mM Tris–HCl buffer, pH 6.8, containing 2.6% (w/v) SDS, 0.5 mM EGTA, 0.5 mM EDTA, 12.6% (w/v) glycerol. Gels were stained with silver (Blum et al., 1986).