Butyl-paraben (BP) is one of the most widely used preservatives in numerous foodstuffs, skin care products, and a variety of drugs, and trypsin is the main digestive enzyme, the research on the bind- ing between the two is essential for human health. In the present paper, the effect of BP on trypsin has been explored using experimental and computational techniques to evaluate BP toxicity at the protein level. The obtained results from molecular docking and kinetic assay revealed BP was embedded in the hydrophobic cavity-S1 binding pocket of the enzyme to inhibit its activity by a com- petitive model. Intrinsic fluorescence of trypsin after interaction with BP revealed the static mode of quenching. FRET indicated that the distance of the enzyme to BP is 1.89 nm with high energy effi- ciency. Thermodynamic results proved that BP spontaneously bound to trypsin in an enthalpy-driven manner, the van der Waals interactions and H-bonds serving as the predominant forces in binding processes. CD spectroscopy and molecular dynamics (MD) simulation revealed that the trypsin struc- ture transformed from the b-Sheet structure to the unordered Coil structure upon interacting with BP. Resonance light scattering (RLS), synchronous fluorescence, and three-dimensional (3 D) spectroscopies further supported the alteration in the conformation of trypsin. Differential scanning calorimetry (DSC) showed that trypsin was somewhat destabilized in the presence of BP. Accordingly, all of the experi- mental data were confirmed by MD simulation.

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