ANTI-NUTRITIONAL CONSTITUENT OF COLOCASIA ESCULENTA ...

The mixture was stirred continuously for two hours. The residue was re-extracted and the
combined filtrate centrifuged at 12 000 g for 20 minutes. The precipitate was discarded and
the crude extract supematant was subjected to 80% (NI4)zS04 saturation and left overnight at
4 DC. Precipitated protein was recovered after centrifugation (12 000 g x 20 min) and the
protein pellet redissolved in a minimum volume of phosphate buffer (0.02 M, pH6.9,
containing 0.3 M NaCl). The protein suspension was transferred to dialysis tubing and
dialysed against the buffer (designated the annnonium sulphate extract). The dialysed sample
was centrifuged and the fraction was analysed for amylase inlnbitor activity.
B.B1.2 Ion-exchange chromatography
The dialysed material, dissolved in 0.02 M phosphate buffer (pH6.9), was loaded on a
column (6 x 1.1 cm) ofDEAE-Sephacel, equilibrated with the same buffer. The column
was eluted with a gradient of (}-().5 M NaCl at a flow rate of 20 00 h- 1 . Four inhibitors,
containing fractions eluted from the column, were pooled and analysed for protein and AI
activity. The absorbance was measured at 280 nm and the graph plotted against tube
numbers.
B.Bl.3 Gel chromatography
The ultimate purification procedure was performed by chromatography on Sephadex G-l00.
During chromatography, the major peaks retained from ion-exchange dissolved in
phosphate buffer were loaded on to a Sephadex G-IOO column (35 x I.I cm), equilibrated
with the same buffer. The elution was performed at a flow rate of 15 00 h- I , fractions were
pooled and analysed for protein and AI activity. Fractions (A-1 and B-2) with AI activities
were collected, dialysed extensively, freeze-dried and dissolved in deionized water.
B.Bl.4 Molecular weight determination by gel filtration chromatography
The relative molecular weight (M,) of the native enzyme was determined by using a
Sephadex G-100 column. Elution was carried out at the flow rate of 15 00 h- I , with an elution
buffer comprising 50 mM tris-HCI pH 7.5. The calIbration curve was constructed using
protein markers: 2 mg 00- 1 cytochrome c (12 400), 3 mg 00- 1 carbonic anhydrase (29 000), 5
mg 00- 1 alcohol dehydrogenase (150 000) and 4 mg 00- 1 13-amylase (200000). 2 mg ml- J of
252