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 alpha-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.
Sharma
Sandeep
S
Department of Biochemistry, Faculty of Life, Sciences, Aligarh Muslim University, Aligarh-202002 (U.P.), India.
Rashid
Fouzia
F
Bano
Bilqees
B
D002942:Circular Dichroism; D015891:Cystatins / Q000737:chemistry* / Q000302:isolation & purification; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D027805:Phaseolus / Q000737:chemistry*; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D014508:Urea / Q000494:pharmacology*
D015891:Cystatins; D014508:Urea