Журнал хроматографии и методов разделения
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Purification and Biochemical Characterization of a Cystatin-Like Thiol Proteinase Inhibitor from Cicer arietinum (Chickpea)

Sheraz AB, Waseem FB, Aaliya S, Shahnawaz KM, Rizwan HK and Bilqees B*

Cystatins are thiol proteinase inhibitors ubiquitously present in many plants as well as animal tissues. In the present study, a cystatin like a thiol proteinase inhibitor was isolated from chickpea using ammonium sulfate fractionation (40-60%) and gel filtration chromatography on a Sephacryl S-100 HR column with 21.09% yield and fold purification of 78.89. It gave a molecular mass of about 25.3 kDa as determined by SDS-PAGE, Dynamic Light Scattering (DLS), and from its gel filtration behavior. The inhibitor was found to have very low carbohydrate content (0.34%) and meager amount (1.45%) of sulfhydryl content. The Stokes radius, frictional, diffusion and sedimentation coefficients of CPC were 4.299 × 10^-8 g/s, 22.82 Å, 12.68 × 10^-7 cm² s^-1 and 2.64 S, respectively. Kinetic analysis of CPC with thiol proteinases (papain, ficin, bromelain) revealed the reversible and competitive mode of inhibition with CPC showing the highest inhibitory efficiency against papain (Ki=0.82 nM) followed by ficin (Ki=17.6 nM) and then bromelain (Ki=41.3 nM). CPC possessed about 25.34% α-helical content as assessed by CD spectroscopy. UV-Visible and fluorescence spectra indicated that upon formation of papain-CPC complex there is a significant conformational change in one or both the proteins of the complex.