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 degrees 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 mu M. respectively. However, it did not exhibit antifungal, antibacterial or mitogenic activity. (C) 2008 Elsevier Ltd. All rights reserved.