Isolation of the lectin and an L4 isolectin from Phaseolus vulgaris by affinity chromatography on insoluble ovomucoid

ABSTRACT

Lectins from extracts of Phaseolus vulgaris seeds have potent cell-agglutinating and lymphocyte-stimulating activity. An affinity adsorbent for lectins with specificity for the oligosaccharide structure was prpared by transforming ovomucoid, an oligosaccharide-rich glycoprotein, into an insoluble and stable gel. The ovomucoid was made insoluble by boiling a 20 per cent solution (200 mg/ml) in 0.1 M Tris-HCl, pH 8.9, for 20 min. This insoluble gel was desialylated by treatment with 50 mN sulfuric acid for 1h at 90ºC and fixed with 1 per cent glutaraldehyde, pH 7,4, for 10 min. The Phaseólus lectin and the L4 isolectin could be isolated essentially in a single-step procedure, using different eluting conditions 50 mM sodium formate buffer, pH 3,0, was used for PHA elutions; a different column was eluted with 15 mM sodium tetraborate, pH 8.0, for desorbed L4 isolectin. Polyacrylamide gel electrophoresis of the lectin showed five distinct bands, whereas the L4 isolectin only presented one band. From 250 mg of saturated column, 8.25 mg of PHA was isolated. This adsorbent could be used several times with little change in binding capacity or selectivity