The sequential extraction of leaf flour proteins was carried by
the Osborne and Voogt (1978) procedure with some modifications.
The defatted flour was dispersed in water (pH 7.0, ratio 1:30, w:v),
subjected to mechanical stirring (60 min) and centrifuged
(15,000 rpm for 40 min). The supernatant, after extensive dialysis
against distilled water in appropriate membranes (pore size
around 10 KDa) for 24–36 h, was centrifuged again, resulting in
albumins (supernatant) and part of the globulins (precipitated),
and the residue was submitted to re-extraction under the same
conditions. The precipitated residue was extracted with 0.5 M NaCl
(pH 7.0, ratio 1:30, w:v) and after centrifugation, the supernatant
was dialyzed as described above and centrifuged again, resulting
in another portion of precipitated globulins, which was added to
the first. The residue from NaCl extraction was successively extracted
with 70% ethanol and 0.1N NaOH to separate prolamin
and glutelin fractions, respectively, from the final residue. The percentages
of protein extracted at each stage were calculated with
respect to the total protein content in defatted flour and analyzed
by nitrogen determination.