Protease inhibitors (PIs) and hemagglutinins are defense proteins produced by many organisms. From Chinese mini-black soybeans, a 17.5-kDa PI was isolated using chromatography on Q-Sepharose, SP-Sepharose, and DEAE-cellulose. A 25-kDa hemagglutinin was purified similarly, but using Superdex 75 instead of DEAE-cellulose in the final step. The PI inhibited trypsin and chymotrypsin (IC50 = 7.2 and 8.8 μM). Its trypsin inhibitory activity was stable from pH 2 to pH 13 and from 0°C to 70°C. The hemagglutinin activity of the hemagglutinin was stable from pH 2 to pH 13 and from 0°C to 75°C. The results indicated that both PI and hemagglutinin were relatively thermostable and pH-stable. The trypsin inhibitory activity was inhibited by dithiothreitol, signifying the importance of the disulfide bond to the activity. The hemagglutinating activity was inhibited most potently by D (+)-raffinose and N-acetyl-D-galactosamine, suggesting that the hemagglutinin was specific for these two sugars. Both PI and hemagglutinin inhibited HIV-1 reverse transcriptase (IC50 = 3.2 and 5.5 μM), proliferation of breast cancer cells (IC50 = 9.7 and 3.5 μM), and hepatoma cells (IC50 = 35 and 6.2 μM), with relatively high potencies
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