Separation of isolectins by high-performance hydrophobic interaction chromatography

High-performance hydrophobic interaction chromatography (HP-HIC) was found to be an effective method for the separation of lectins into isolectin fractions. All of the purified lectins used in this study, Phaseolus vulgaris haemagglutinin (PHA), wheat germ agglutinin (WGA), Ricinus communis agglutinin (RCA), and Arachis hypogaea agglutinin (AHA), were prepared by affinity chromatography. HP-HIC was performed on a column (15 X 2.1 cm) of TSK gel Phenyl-5PW at room temperature. The lectin sample, dissolved in 1.0 or 0.5 M ammonium sulphate in phosphate buffered saline (pH 7.4) (PBS), was applied to the column and eluted with a linear gradient from 1.0 or 0.5 M ammonium sulphate in PBS to 0 M ammonium sulphate in PBS at a flow-rate of 4 ml/min. In the case of RCA, addition of glycerol to the elution buffer resulted in sharper isolectin peaks. PHA, WGA, RCA, and AHA were rapidly separated into 5, 5, 4, and 6 isolectins, respectively.