Comprehensive insights into the binding of crystal violet and pepsin: Spectroscopic analysis, molecular model and biochemical method

•CV quenched the intrinsic fluorescence of pepsin in a combined mechanism.•The conformational change of pepsin was induced by CV.•Electrostatic interaction was nonnegligible for the stability of CV-pepsin binding process.•Tyr75, Tyr189 and Asp215 residues made a great contribution towards the binding process.•The activity of pepsin was activated by CV. As a well-known triphenylmethane dye, crystal violet (CV) has received extensive concerns owing to its harmful influence on human health. Pepsin is a digestive enzyme in the stomach that plays a crucial part in several physiological and pathological process. This research aimed to use spectroscopic experiments, computer simulation and enzyme activity assays to investigate the influence of CV on the functions and structure of pepsin from the perspective of binding. Fluorescence and UV–vis spectra confirmed the complex formation between CV and pepsin. Ka was in the order of 104 M−1, implying a moderate affinity between CV and pepsin. Multi non-covalent interactions played a crucial part in this binding process. The conformational alterations of pepsin caused by CV were reflected in β-sheet content reducing, and the presence of CV caused an activation of pepsin. The outcomes of molecular docking indicated that the interaction of Tyr75/Tyr189 with CV is the major cause for the fluorescence quenching phenomenon of pepsin. For molecular dynamics (MD) simulation, the CV-pepsin and free pepsin systems reached equilibrium in 40 ns. In addition, the compactness of pepsin changed after interacting with CV, which was in line with the results of solvent experiment. From the above results, CV has effects on the properties of pepsin, which may provide novel insights into the toxicological study of CV. [Display omitted]