A stable trypsin inhibitor from Chinese dull black soybeans with potentially exploitable activities

A dimeric 40-kDa Kunitz-type trypsin inhibitor was isolated from seeds of the Chinese black soybean Glycine max cv. 'Dull Black'. The purification protocol comprised ion exchange chromatography on Q-Sepharose, SP-Sepharose, and Mono Q, and gel filtration on Superdex 75. The trypsin inhibitor inhibited chymotrypsin, albeit to a lesser extent than it inhibited trypsin. Its trypsin-inhibitory activity was unaffected after exposure to pH 1-14, or to temperatures up to 80 °C. The trypsin inhibitor was inhibited by dithiothreitol in a dose-dependent (from 2.5 to 50 mM) and a time-dependent (from 5 to 120 min) manner. Besides inhibiting serine proteases, the trypsin inhibitor demonstrated additional biological activities including stimulation of nitric oxide production by macrophages. It inhibited HIV-1 reverse transcriptase, cell-free translation and proliferation of liver cancer cells and breast cancer cells, with an IC₅₀ value 9.4, 14, 39 and 70 μM, respectively. However, it did not exhibit antifungal, antibacterial or mitogenic activity.