Azo-dyes, a diverse group of synthetic dyes, are commonly used for coloring food products. However, these dyes are persistent in nature with the potential to cause harmful effects on the human body. Hence, it seems that studies are required about the effects of azo dyes on the structure and function of biomacromolecules. Ponceau 4R (P4R) is an azo-dye found in foods, beverages, and pharmaceuticals, but its potential risk on gastrointestinal enzymes such as trypsin is poorly characterized. Thus, this study focused on analyzing the binding behavior between trypsin and P4R by spectroscopic, kinetic, and theoretical methods. Intrinsic fluorescence revealed that there was an interaction between P4R and trypsin, and P4R could quench the trypsin fluorescence by a static model. The resonance light scattering (RLS) method was used to confirm the formation of the P4R-trypsin complex. The thermodynamic data indicated that the predominant interactions between P4R and trypsin were Van der Waals and hydrogen bonds, consistent with docking results. The distance between P4R and trypsin after the interaction was calculated based on fluorescence resonance energy transfer (FRET) theory and found to be 3.36 nm. Synchronous fluorescence, three-dimensional fluorescence, UV–Visible spectrophotometry, and circular dichroism (CD) analyses indicated that the conformation of trypsin was changed upon binding with P4R, which could affect the trypsin function. Kinetic studies revealed that P4R inhibited trypsin activity in a non-competitive model, and docking analysis confirmed that P4R was not bound to the active site of the trypsin. The molecular dynamics (MD) simulation studies showed that the root mean square deviation (RMSD) in the trypsin-P4R complex (0.270 ± 0.02 nm) was larger than that of the free trypsin system (0.221 ± 0.01 nm), which agreed with CD and thermal stability experiments. Thus, P4R was shown to be a potent inhibitor and quencher of trypsin.

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