Isolation of the lectin and an L4 isolectin from Phaseolus vulgaris by affinity chromatography on insoluble ovomucoid.
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 prepared by transforming ovomucoid, an oligosaccharide-rich glycoprotein, into an insoluble and stable gel. The ovomucoid was made insoluble by boiling a 20% 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 1 h at 90 degrees C and fixed with 1% glutaraldehyde, pH 7.4, for 10 min. The Phaseolus 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 elution; 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.
Gonçalves
R B
RB
Departamento de Diagnóstico Oral, Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas, Piracicaba, SP, Brasil.
Costa
C P
CP
D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000737:chemistry*; D037102:Lectins / Q000302:isolation & purification*; D010835:Phytohemagglutinins / Q000302:isolation & purification*; D037121:Plant Lectins; D010946:Plants, Medicinal
D037102:Lectins; D010835:Phytohemagglutinins; D037121:Plant Lectins