Interactions between food additive butylated Hydroxytoluene (BHT)/its metabolites and pepsin: Mechanisms and biological implications.

Butylated hydroxytoluene (BHT) is a synthetic phenolic antioxidant extensively used in food. Given the increasing biosafety concerns regarding BHT and its metabolites (BHT-OH and BHT-COOH), their interactions with pepsin were systematically investigated using multispectral techniques, molecular docking, and simulation methods. Fluorescence analysis indicated static quenching and stable complex formation, with binding affinities following the order BHT-OH > BHT-COOH > BHT. Hydrogen bonding and van der Waals forces dominated the interactions, and the carboxyl group of BHT-COOH contributed additional hydrogen bonds. Binding induced conformational changes in pepsin, altered the microenvironment of tryptophan residues, and resulted in dose-dependent inhibition of enzymatic activity. Meanwhile, the binding also significantly reduced the radical scavenging and reducing capacity of BHT and its metabolites. These findings highlight the critical role of functional group substitution in modulating gastrointestinal interactions and potential health risks of these compounds.