Identification of an Independent Measurement Method for Denaturation Studies of Cytochrome C

摘要

Developing some substitutionary ways such as electrochemical methods might be useful in order tofollow structural changes of proteins. In this study, the effect of denaturants such as Urea and SDS(sodium dodecyl sulfate) was investigated on Cytochrome C structure, a metalloproteinase with anoxide/redox property. Structural alterations were studied with potentiometer and different spectrometertechniques. Cytochrome C was prepared in 20mM phosphate buffer with pH=7.0. The protein wastreated with different concentrations of Urea and SDS. Related absorbance of native and denaturedprotein were recorded with Cary 50 (UV-Visible) and AVIV-215 (Circular Dichroism)spectrophotometers in range of 200-700 nm. Electrochemical measurements were also recorded withan acetate/Galvan acetate potentiometer device. Cytochrome C was reported to be structurally stabilein 5-8 and 3-9.5mM concentrations of Urea, which were monitored by voltammetry and spectroscopymethods, respectively. The protein was active in high concentration (26mM) of SDS. Theconcentration of SDS 20mM seems to be sufficient for proper folding of 0.5mM Cytochrome C. It hasbeen concluded that, 40 M Cytochrome C, crudity, and etc do not impact voltammetry results and itis an independent method for detection of structural changes of proteins. Voltammetry method is moresensitive to small changes in protein structure compared with spectroscopy methods.