Spectrophotometer measurements to characterize conformational state of the proteins: P53 analysis

The development of sensing techniques for the analysis of protein samples represents an area of great interest for biotechnology, pharmacology and diagnostics. Traditional biochemical assays available present limitations in term time and cost efficiency. On the contrary, optical based techniques, such as spectrophotometry, might offer the interesting possibility of a non-invasive and continuous monitoring. The present work proposes a spectrophotometer-based method of identifying different conformational states of p53, a redox sensitive protein involved in several pathophysiological processes. Samples containing three different structural states of p53 (Wild type p53, Denatured p53 and Oxidized p53) has been investigated using spectrophotometer in a measuring wavelength range from 185nm to 1400nm, in order to detect the differences in light absorption. The unfolding state of p53 redox products has been further studied performing a label-based silver stripping voltammetry. Results from the spectrophotometric analysis showed different absorbance peaks at distinct wavelength for each conformation, suggesting the possibility to use this technique to discriminate the different p53 redox products tested. Further, these results appeared to be well supported from the binding-affinity label-based test. Overall the study allowed to state the possibility to identify p53 different conformational states through this simple and non-invasive method, thus reducing the complexity of the procedures involved in conventional methods.