Luminescence Insights to Binding and Conformational Analysis of Graphene Oxide - Pepsin system: A Multi-Spectroscopic and In-Silico Approach.

Pepsin is a key proteolytic enzyme involved in the gastric digestion. Graphene oxide (GO) despite the promising biomedical applications, lacks consensus for its use in medicines. One of the leading causes in this respect is the limited understanding of its interaction with model proteins. In this background, the present study investigates the biophysical interaction of GO with pepsin using UV-Vis, Fluorescence, FT-IR spectroscopy, and in-silico modelling. UV-Vis spectra of pepsin revealed slight hypsochromic and hyperchromic shift in the presence of GO indicating alteration in pepsin's structure corroborated by fluorescence quenching study. Further analysis of fluorescence data shows a decrease in Stern-Volmer quenching constant, K_sv (order of 10⁵ L g^-1) decreases with increasing temperature that indicates static quenching which is essentially due to complexation. The complexation was further characterized in terms of association constant (non-covalent interactions) and the equivalent number of binding sites (close to one). Thermodynamic analysis suggested the binding interaction is essentially entropy driven. Synchronous fluorescence spectra suggested microenvironment changes for both the tryptophan and tyrosine residues in the binding sites. Detailed secondary structure study of pepsin (native and in presence of GO) using ATR-FTIR confirmed significant changes in the protein conformation (α-sheet, random coil, and β-helix) upon interaction with GO. Molecular docking suggested the involvement of H-bonding and π- π interactions within the GO-pepsin system with a binding score of -11.47 kcal/mol. DLS study indicated concentration dependent increase in hydrodynamic diameter of pepsin in presence of GO. Finally, physiological activity of pepsin was found decreased (up to 54%) upon incubation with GO suggested change in its activity. This work provides valuable insights into the binding mechanism and interaction of GO with pepsin with potential implications for the development of an oral GO based formulations in the near future.