Comparison of the phytohaemagglutinin from red kidney bean (Phaseolus vulgaris) purified by different affinity chromatography

Affinity chromatography (AC) on Affi-Gel blue gel column and thyroglobulin (Tg)-Sepharose 4B column, respectively, were compared for their efficiency in purifying phytohaemagglutinin (PHA) from red kidney beans (Phaseolus vulgaris). Considering the purity and haemagglutinating activity of the obtained samples, Affi-Gel blue gel exhibited less affinity for PHA than Tg-Sepharose matrix. Affi-Gel blue purified sample showed multiple bands in SDS-PAGE gel, which further confirmed that Affi-Gel blue bound non-PHA proteins as well as PHA. PHA purified by one-step Tg-Sepharose column gave significantly (p < 0.05) higher purity (0.75 ± 0.13 mg PHA/mg lyophilized powder) than the sample purified by two-step (Affi-Gel blue first and then Tg-Sepharose) purification (0.62 ± 0.20 mg PHA/mg lyophilized powder). Circular dichroism (CD) spectra showed that the sample purified by one-step Tg-Sepharose column had similar secondary structures with the sample purified by two-step purification. Thus, one-step Tg-Sepharose purification was effective and time-saving for the preparation of PHA and a promising substitute for the two-step purification method.