Phytohemagglutinin isolectins extracted and purified from red kidney beans and its cytotoxicity on human H9 lymphoma cell line

Abstract

Five isolectins, L 4, L 3E 1, L 2E 2, L 1E 3 and E 4, were purified from red kidney bean ( Phaseolus vulgaris) by affinity chromatography on a porcine thyroglobulin bound Sepharose ® 4B column, and subsequent separation by Sulfopropyl Sephadex C-50 ion exchange chromatography. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), one clear band appeared for each isolectin, which confirmed the high purity. The decreased haemagglutination activity in an order of E 4, L 1E 3, L 2E 2, L 3E 1 and L 4 further proved the high separation efficiency of the two columns. Low concentration (1 μg/mL) of E 4 was able to stimulate the proliferation of human H9 lymphoma cells but strong cytotoxicity was observed at higher concentration. The H9 cell viability was inhibited by the five isolectins in a dose-dependent manner. The concentrations that inhibited 50% of the H9 cell viability were determined to be 3.385 μM for L 4, 7.778 μM for L 3E 1, 9.939 μM for L 2E 2, 20.840 μM for L 1E 3 and 33.004 μM for E 4, respectively. L 4 showed more potent cytotoxicity than E 4, which was probably due to stronger binding of L 4 with the cell-surface carbohydrates than E 4. It may also be due to the partial masking of E 4 cytotoxicity by the glycoprotein supplemented in the medium.