Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/20/2004
Publication Date: 7/3/2004
Citation: Bawadi, H.A., Antunes, T.M., Shih, F.F., Losso, J.N. 2004. In vitro inhibition of the activation of pro-matrix metalloproteinase 1 (pro-mmp-1) and pro-matrix metalloproteinase 9 (pro-mmp-9) by rice and soybean bowman - birk inhibitors. Journal of Agricultural and Food Chemistry. Interpretive Summary: Degenerative diseases, such as arthritis, cancers, and AIDS complications, are often associated with angiogenesis or the formation of new blood vessels from pre-existing vascular ones. Angiogenesis is catalyzed by matrix metalloproteinases (MMPs), which in turn, are activated by proteases. Therefore, by controlling the activation of MMPs, protease inhibitors could be useful in resisting the onset and/or progression of diseases. This research investigated the inhibitory activity of Bowman-Birk inhibitors from rice and soybean (rBBI and sBBI) against the activation of several MMPs. The results confirmed and clarified conditions for these protease inhibitors as effective agents in controlling the activity of MMPs, and the occurrence of angiogenesis. The research indicates that inhibitors rBBI and sBBI have the potential as health-enhancing functional food ingredients.
Technical Abstract: The in vitro inhibitory activity of the rice Bowman-Birk inhibitor (rBBI), or soybean Bowman-Birk inhibitor (sBBI), against trypsin-catalyzed activation of pro-matrix metallogroteinase 1 or 9 (pro-MMP-1 or pro-MMP-9), respectively, was investigated using electrophoresis with silver staining, heparin-enhanced zymopraphy, biothnylatede gelatin, Biotrrak assay, and fluorescence quenched substrate hydrolysis. rBBI at concentrations of 0.08-0.352 mg/mL dose-dependently inhibited the in vitro activation of 45 ug/mL pro-MMP-1 by trypsin. Heparin-enhanced zymopgraphy analysis of pro-MMP-1, trypsin-activated MMP-1, and a mixture of pro-MMP-1-trypsin-rBBI showed clear zones associated with trypsin-activated MMP-1, and the absence of clean zones in lanes containing pro-MMP-1 or a mixture of pro-MMP-1, trypsin, and rBBI. The results of the Biotrak assay also indicated that rBBI dose-dependently suppressed the activation of pro-MMP-1 by trypsin. sBBI dose-dependently inhibited the activation of 100 ug/mL of pro-MMP-9 in the presence of trypsin and BBI did not hydrolyze gelatin, whereas p-aminophenylmercury acetate (APMA)-activated MMP-9 and trypsin-activated MMP-9 caused significant hydrolysis of gelatin. Quenched fluorescence substrate hydrolysis for total MMP activity showed that pro-MMP-1 or pro-MMP-9 did not hydrolyze the substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2; active MMP-1 or MMP-9 hydrolyzed the substrate, but lower substrate hydrolysis was obtained when pro-MMP-1 or MMP-9 was incubated with trypsin in the presence of increasing concentrations of rBBI. The results are discussed in light of the role of MMP-1 and MMP-9 in the process of angiogenesis, and the potential of rBBI or sBBI as a functional food ingredient.