Individual mono and co-interactions of butylated hydroxytoluene and its metabolite with pepsin: Multi-pronged research strategies.

In this study, the interactions between butylated hydroxytoluene (BHT) and its metabolite 2,6-Di-tert-butyl-p-benzoquinone (BHT-Q) with pepsin (PEP) were explored using multispectral measurements and computer prediction techniques. UV-vis absorption spectra, fluorescence lifetime, and Stern-Volmer quenching analysis showed static fluorescence quenching of PEP by BHT/BHT-Q. Negative thermodynamic parameters indicated that the spontaneous formation of complexes was primarily driven by van der Waals (vdW) forces and hydrogen bonds (HB). Synchronous fluorescence and circular dichroism spectroscopy revealed conformational changes induced by BHT/BHT-Q on PEP. Furthermore, BHT and BHT-Q inhibited PEP's enzymatic activity, while PEP suppressed their antioxidant activity. Interestingly, BHT-Q weakened BHT's binding strength to PEP, affecting the enzyme inhibition rate. Computer predictions highlighted the integral role of hydrophobic interactions. Moreover, BHT and BHT-Q exhibited different effects on the stability and compactness of PEP, the residue environment of PEP became more flexible or rigid in the presence of BHT and BHT-Q. Changes in the hydrophobic solvent accessible surface area (SASA) elucidated that the microenvironment of hydrophobic residues of PEP was changed after binding with BHT and BHT-Q. Ultimately, BHT's stronger binding affinity to PEP than BHT-Q was attributed mainly to its larger negative surface area, facilitating interactions with more amino acid residues.