Multimeric Association of Purified Novel Bowman-Birk Inhibitor From the Medicinal Forage Legume Mucuna pruriens (L.) DC.

A Bowman–Birk protease, M. pruriens trypsin inhibitor (MPTI) was purified from the seeds by 55.702 fold, and revealed a single trypsin inhibitor on a zymogram with a specific activity of 202.31 TIU/mg of protein. On SDS–-PAGE under non-reducing conditions, the protease trypsin inhibitor fraction (TINR) exhibited molecular weights of 74 kDa and 37 kDa, and under reducing conditions (TIR) - 37 kDa and 18 kDa. TINR-37 revealed protease inhibitor activity on native PAGE and 37 kDa and 18 kDa protein bands on SDS-PAGE. TINR-74 showed peaks corresponding to 18.695, 37.39, 55.85, and 74.78 kDa on UPLC coupled with ESI/QTOF-MS. Similarly, TINR-37 displayed 18.695 and 37.39 kDa peaks. Further, TIR-37 and TIR-18 exhibited peaks corresponding to 37.39 and 18.695 kDa. Multiple peaks observed by UPLC-ESI/QTOF analysis revealed the multimeric association, confirming the characteristic and functional features of BBIs. The multimeric association helps to achieve more stability, thus enhancing their functional efficiency. MPTI was found to be a competitive inhibitor which again suggested that it belongs to BBI family of inhibitors, displayed an inhibitor constant of 1.3 × 10-6 M and further demonstrates potent anti-inflammatory activity. The study provided a comprehensive basis of the identification of multimeric associates and their therapeutic potential, which could elaborate the stability and functional efficiency of the MPTI in native state from M. pruriens.