Trypsin Isoinhibitors with Antiproliferative Activity toward Leukemia Cells from Phaseolus vulgaris cv “White Cloud Bean”

A purification protocol that comprised ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose, and gel filtration by FPLC on Superdex 75 was complied to isolate two trypsin inhibitors from Phaseolus vulgaris cv “White Cloud Bean”. Both trypsin inhibitors exhibited a molecular mass of 16 kDa and reduced the activity of trypsin with an IC(50) value of about 0.6 μM. Dithiothreitol attenuated the trypsin inhibitory activity, signifying that an intact disulfide bond is indispensable to the activity. [Methyl-(3)H] thymidine incorporation by leukemia L1210 cells was inhibited with an IC(50) value of 28.8 μM and 21.5 μM, respectively. They were lacking in activity toward lymphoma MBL2 cells and inhibitory effect on HIV-1 reverse transcriptase and fungal growth when tested up to 100 μM.