Isolation and Characterization of a Kunitz-Type Trypsin Inhibitor with Antiproliferative Activity from Gymnocladus Chinensis (Yunnan Bean) Seeds

Abstract

A 20-kDa Kunitz-type trypsin inhibitor was isolated from Gymnocladus chinensis (Yunnan bean) seeds. The isolation procedure involved ion exchange chromatography on diethylaminoethyl cellulose (DEAE-cellulose), affinity chromatography on Affi-gel blue gel, ion exchange chromatography on sulfopropyl sepharose (SP-sepharose), and gel filtration by FPLC on Superdex 75. The trypsin inhibitor was adsorbed on DEAE-cellulose, unadsorbed on Affi-gel blue gel, and adsorbed on SP-Sepharose. It dose-dependently inhibited trypsin with an IC50 value of 0.4 μM. Dithiothreitol reduced its trypsin inhibitory activity, suggesting that an intact disulfide bond is indispensable to the activity. It suppressed [methyl-3H] thymidine incorporation by leukemia L1210 cells and lymphoma MBL2 cells with an IC50 value of 4.7 and 9.4 μM, respectively. There was no effect on human immunodeficiency virus4-1 reverse transcriptase activity and fungal growth when the trypsin inhibitor was tested up to 100 μM.