The crude extract was incubated for 30 min at 45 C and centrifuged
at 10,000g for 10 min at 4 C. The supernatant (300 mL) obtained
was then fractionated with iced ethanol by adapted
methodology of Cortez and Pessoa (1999). The ethanol concentration
values employed in the precipitation experiments were 0–30%
(Fraction F1) and 30–70% (Fraction F2). For each fraction, the ethanol
was slowly added to the extract under agitation (200 rpm) at
4 C with a burette in a cold chamber. After the addition of ethanol,
the agitation was stopped for 2 h and the mixture was centrifuged
at 10,000g for 15 min at 4 C. The pellets (Fraction F1 and F2) were
dissolved in 75 mL 0.1 M Tris–HCl buffer (pH 8.0) at 25 C and dialyzed
against 4 L of 0.05 M Tris–HCl buffer (pH 8.0) for 2 h. After
this period, the buffer was renewed and dialysis was repeated for
a further 2 h. Finally, after a third renewal of the buffer, dialysis
was allowed to proceed overnight.