Soybean seeds contain three groups (A, C, and D) of Bowman-Birk inhibitors (BBIs). In this study, highly purified BBI-A (approximately 96%) was obtained from soybean whey at the 0.1 g level by the complex coacervation method. BBI-A has seven disulfide bonds (SS) and no sulfhydryl group and exhibits trypsin inhibitor activity (TIA) and chymotrypsin inhibitor activity (CIA). The X-ray structure has shown that BBI-A has five exposed SS and two buried SS. Because of steric hindrance, it was reasonable to consider that dithiothreitol first attacks the five exposed SS and then the two buried SS, which was supported by the results that SS reduction with dithiothreitol could be divided into quick and slow stages, and the critical point was close to 5/7. The effects of SS reduction on TIA and CIA could be divided into three stages: when one exposed SS was reduced, both TIA and CIA decreased to approximately 60%; with further reduction of exposed SS, CIA gradually decreased to 8% and TIA gradually decreased to 26%; with further reduction of buried SS, CIA gradually decreased to 2% and TIA slightly decreased to 24%. Far-ultraviolet (far-UV) circular dichroism (CD) spectra showed that the secondary structure of BBI-A was slightly changed, whereas near-ultraviolet (near-UV) CD spectra showed that the conformation of BBI-A was substantially changed after the five exposed SS were reduced; further reduction of buried SS affected the conformation to some extent. The results of Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and from a C8 column showed the same trend as near-UV CD spectra. BBI-A has a structural peculiarity in that two hydrophobic patches are exposed to the exterior (in contrast to typical soluble proteins), which was attributed to the seven SS by some researchers. These results support the hypothesis that hydrophobic collapse of the exposed hydrophobic patches into a regular hydrophobic core occurred after the reduction of SS in BBI-A.
Keywords: Bowman-Birk inhibitor; bioactivity; conformational change; disulfide bond.