2 × 42 cm), which was eluted with 0.1 M Tris–HCl, pH 8.0, at a flow rate of 0.34 ml min−1. Each fraction collected was tested for tryptic activity.

The protein peaks with high specific trypsin activity were pooled and applied on a benzamidine-agarose column (1 ml of packed column), which was eluted see more first with Tris–HCl 0.1 M, pH 8.0. Then, it was eluted with 0.05 KCl–HCl M, pH 2.0, and collected in 40 μl of 1.5 M Tris–HCl buffer, pH 9.0. Both benzamidine-agarose steps were carried out at the same flow rate (0.5 ml min−1). Each fraction was tested for tryptic activity. The protein peak with the highest trypsin activity was pooled and, after dialysis against 0.01 M Tris–HCl buffer, pH 8.0, it was stored at −25 °C to be used in the characterisation experiments. All steps were analysed by SDS–PAGE. Thirty microlitres of 8 mM N-α-benzoyl-dl-arginine-p-nitroanilide (BApNA), prepared in dimethylsulphoxide (DMSO), was incubated in microtitre wells with the enzyme (30 μl) and 0.1 M Tris–HCl, pH 8.0 (140 μl). The release of p-nitroaniline was measured as an increase in absorbance at 405 nm in a microplate reader (BioRad Model X-Mark™, USA). Controls were performed without enzyme. One unit of enzyme activity is considered

as the amount of enzyme able to produce 1 μmol of p-nitroaniline per minute. Protein content was estimated Z-VAD-FMK price by measuring sample absorbance at 280 nm and 260 nm, using the following equation: [protein] mg/ml = 1.5 × A280nm − 0.75 × A260nm (Warburg & Christian, 1941). SDS–PAGE was carried out according to the method described by Laemmli (1970), using a 4% (w/v) stacking gel and a 12.5% (w/v) separating gel. Lyophilised samples from the affinity Protein tyrosine phosphatase chromatography pool (50 μg of protein) and a molecular mass standard were added to a solution containing 10 mM Tris–HCl (pH 8.0), 2.5% SDS, 10% glycerol,

5% β-mercaptoethanol and 0.002% bromophenol blue, heated at 100 °C for 3 min and applied onto the electrophoresis gel. The electrophoretic running was conducted at variable voltage and constant current conditions. After running, the gel was stained for protein in a solution containing 0.25% (w/v) Coomassie Brilliant Blue, 10% (v/v) acetic acid and 25% methanol, for 30 min. The background of the gel was destained by washing in a solution containing 10% (v/v) acetic acid and 25% methanol (v/v). The molecular weights of the protein bands were estimated using the 198–6.8 kDa molecular mass protein standards (Bio-Rad laboratories). The assay was carried out using BApNA as a substrate in the range of final concentration from 0.025 to 2 mM) and under the same conditions (pH 8.0 and 25 °C) as described above. The reaction (triplicates) was initiated by adding 30 μl of purified enzyme solution (21.3 μg of protein/ml). Reaction rates were fitted to Michaelis–Menten kinetics, using Origin 6.0 Professional. The influences of both temperature and pH on trypsin activity of the A.