Redox status of Bowman-Birk inhibitor from soybean influence its in vitro antioxidant activities

Soybean (Glycine max) is a major protein source for animal and human nutrition. The Bowman-Birk protease inhibitor (BBI), ranking 3rd of protein contents among soybean seed storage proteins, is a major antinutritional factor. BBI was incubated with 1 mM DTT at 37°C for 2 h and loaded directly onto a Sephadex G-25 gel column for purification. The molecular mass of the reduced form of BBI is ca. 8 kDa determined by SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel electrophoresis). The methodologyb we used includes total antioxidant status, (1,1-diphenyl-2-picryl hydrazyl) DPPH staining, DPPH radical scavenging activity, reducing power method, Fe²⁺-chelating ability, FTC (ferric thiocyanate) method, and protection calf thymus DNA against hydroxyl radical-induced damage. The oxidized and reduced form of BBI with a concentration of 200 μg/mL exhibited the highest activity (expressed as 4.74 ± 0.36 and 7.20 ± 0.20 mM Trolox equivalent antioxidative value, TEAC) in total antioxidant status test. In the DPPH staining the reduced form of BBI appeared as white spots when it was diluted to 12.5 μg/mL (a final amount of 0.6 μg). Like total antioxidant status, the reducing power, Fe²⁺-chelating ability, FTC activity and protection against hydroxyl radical-induced calf thymus DNA damage all showed that the reduced BBI exhibited higher antioxidative activities than the oxidized BBI. The results suggested that the reduced BBI exhibited higher antioxidative activities than the oxidized BBI in a series of in vitro tests. These findings provide one of the molecular bases for BBI applications to treat various serious diseases.