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

Five isolectins, L4, L3E1, L2E2, L1E3 and E4, 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 E4, L1E3, L2E2, L3E1 and L4 further proved the high separation efficiency of the two columns. Low concentration (1 μg/mL) of E4 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 L4, 7.778 μM for L3E1, 9.939 μM for L2E2, 20.840 μM for L1E3 and 33.004 μM for E4, respectively. L4 showed more potent cytotoxicity than E4, which was probably due to stronger binding of L4 with the cell-surface carbohydrates than E4. It may also be due to the partial masking of E4 cytotoxicity by the glycoprotein supplemented in the medium.