Purification, characterization and kinetics of protease inhibitor from fruits of Solanum aculeatissimum Jacq

AbstractDouble-headed protease inhibitor was isolated, purified and characterized including its kinetics from the fruits of Solanum aculeatissimum Jacq. (SAPI). The inhibitor was purified to homogeneity via four sequential step procedure, i.e., salt precipitation to Sepharose affinity chromatography. The purity was confirmed by reverse phase HPLC chromatography. The molecular mass was detected using size elution chromatography (22.2kDa). SAPI inhibits trypsin and chymotrypsin simultaneously and the molar inhibitory ratio for trypsin and chymotrypsin was 1:1, i.e., it functions as double headed PI of Bowman–Birk group. Purified SAPI showed optimal specific activities of 502 and 433.7U/mg with trypsin and chymotrypsin, respectively. Overall, the fold of purification increased with remarkable yield. Native-PAGE showed four isoinhibitors (pI: 4.7, 5.2, 5.6 and 5.9). Dixon plots and Lineweaver–Burk double reciprocal plots revealed competitive mode of inhibition. High pH amplitude (2–12) and broad temperature range (10–80°C) were observed for SAPI. Circular dichroism spectrum of native SAPI displayed random structure with more β-sheets. Further, metal ions, detergents, oxidizing and reducing agents affected the inhibitory potential of SAPI in different levels. Chemical modification studies to analyze the key amino acid present in the reactive site of SAPI revealed the presence of lysine and tryptophan residue(s)