Abstract
A Bowman-Birk protease, i.e., Mucuna 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 sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under non-reducing conditions, the protease trypsin inhibitor fraction [i.e., trypsin inhibitor non-reducing (TINR)] exhibited molecular weights of 74 and 37 kDa, and under reducing conditions [i.e., trypsin inhibitor reducing (TIR)], 37 and 18 kDa. TINR-37 revealed protease inhibitor activity on native PAGE and 37 and 18 kDa protein bands on SDS–PAGE. TINR-74 showed peaks corresponding to 18.695, 37.39, 56.085, and 74.78 kDa on ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization/quadrupole time-of-flight-mass spectrometry (ESI/QTOF-MS). Similarly, TINR-37 displayed 18.695 and 37.39 kDa peaks. Furthermore, TIR-37 and TIR-18 exhibited peaks corresponding to 37.39 and 18.695 kDa. Multiple peaks observed by the UPLC-ESI/QTOF analysis revealed the multimeric association, confirming the characteristic and functional features of Bowman-Birk inhibitors (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 the 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 for the identification of multimeric associates and their therapeutic potential, which could elaborate the stability and functional efficiency of the MPTI in the native state from M. pruriens.
Highlights
Protease inhibitors (PIs) are small proteins or peptides that inhibit the proteolytic activity of enzymes by forming highaffinity stoichiometric complexes (Bode and Huber, 2000)
The trypsin-specific PI was purified to homogeneity from M. pruriens seeds using different conventional techniques such as acid extraction, salt fractionation, CM-cellulose chromatography, Sephadex G-75 gel filtration chromatography, and preparative gel electrophoresis
The crude PI extract was subjected to 0–25% and 25–50% ammonium sulfate fractionation
Summary
Protease inhibitors (PIs) are small proteins or peptides that inhibit the proteolytic activity of enzymes by forming highaffinity stoichiometric complexes (Bode and Huber, 2000). Several types of SPIs have been extensively purified, characterized, and assessed for their biological potential from various sources (Clemente et al, 2019) In plants, these proteins are involved in many physiological processes, such as the regulation of endogenous and exogenous proteolysis, delaying senescence, and acting as defensive molecules, thereby protecting against pathogens (Pak and Van Doorn, 2005; Joshi et al, 2014; Yasin et al, 2014, 2020). These proteins are involved in many physiological processes, such as the regulation of endogenous and exogenous proteolysis, delaying senescence, and acting as defensive molecules, thereby protecting against pathogens (Pak and Van Doorn, 2005; Joshi et al, 2014; Yasin et al, 2014, 2020) They are involved in signal transduction, mobilization of storage proteins, and morphogenesis during plant development (Yasin et al, 2014, 2020; Rustgi et al, 2017)
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