Unfolding During Urea Denaturation of a Low Molecular Weight Phytocystatin (Thiol Protease Inhibitor) Purified from Phaseolus mungo (Urd)

In the present study, two phytocystatins were purified to homogeneity as peaks I and II with molecular weights of 19 kDa and 17 kDa, respectively, as determined by SDS-PAGE and mass spectrometry. Both PMCs I and II were purified with a greater than 1000-fold purification and overall yield of about 16-18%. The effect of urea on PMC I and II was analysed by fluorescence and Circular Dichroism (CD) spectroscopy. Fluorescence studies suggest a red shift of the maximum emission at higher urea concentrations. PMC I and II are extremely stable protein inhibitors with regards to temperature and pH stability. FTIR studies show predominant a-helical structure in both the cystatins. CD analysis results show change in urea concentration-dependent loss in ellipticity, as well as in the shape of the CD spectrum compared to the intact phytocystatin.