ANTI-NUTRITIONAL CONSTITUENT OF COLOCASIA ESCULENTA ...

Dextran blue was used to determine the void volume (Vo). A calibration curve between log­
log molecular weights of protein marlrers and the partition coefficient values (KAV) was
constructed.
B.BI.S Enzyme assay
The a-amylase and a-amylase inlnllitor activity were measured using Bernfeld's method
(Bernfeld, 1955). a-Amylase inhibitor extracts were added to a one-per-eent (w/v) starch
solution in a 20 mM phosphate buffer, containing 0.4 mM NaCl (pH 6.9) and a-amylase.
The mixtures were incubated at 37° C for 30 minutes. Reaction was stopped by the addition
of 10 00 ofdinitrosalicylic acid reagent (DNS). This solution was boiled for five minutes,
then cooled and the absorbance was read at 530 urn. One amylase unit is defined as the
amount of enzyme that will liberate I !1mol of maltose from starch under the assay
conditions (pH 6.9; 37° C; five minutes). Inhibitory activity is expressed as the percentage
ofinlnllited enzyme activity out ofthe total enzyme activity used in the assay.
B.BI.2 Kinetic studies
B.BI.2.1 pH
For all kinetic studies other than pH, both enzymes obtained after gel-filtration on Sephadex
G-IOO were used.
The effect ofpH was checked using the following buffers (0.1 M): sodium acetate (pH 5),
sodium phosphate (pH 6-7), Tris-HCI (pH 8) and glycine-NaOH buffer (pH 9-10).
The AI activity was assayed at different pH values (pH I - pH 10) for five minutes at 3r C and
the remaining amylase activity was determined.
B.BI.2.2 Temperature
The optimum temperature for inhibition ofthe inlnllitor was determined by assaying the amylase
activity at temperatures ranging from 20° C to lOO °c at pH 6.9. Coustant amounts (loo) of AI
were preincubated with phosphate buffer (pH 6.9) for five minutes at 2-100° C. Remaining
amylase activity was determined.
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