Evaluation of the interaction of food dye fast green FCF with the digestive enzyme trypsin

• The interactions of fast green FCF with trypsin were investigated. • FCF could quench the fluorescence of trypsin through static quenching. • The major driving forces are van der Waals force , and hydrogen bonding . • FCF binding to trypsin induces the conformational changes of the enzyme . • Molecular docking studies demonstrate that the FCF interacts effectively with key residues in the active site of the trypsin, Ser 195, His 57 and Gly 219. Fast green FCF (FCF) is used for dyes, cosmetics, drugs, and food. Due to the adverse effects of dyes on human macromolecules, these effects need to be investigated to obtain clear information on the harmful effects of these dyes. Trypsin is one of the main digestive enzymes. Researching the interaction between the two essentials for human health. The effects of the FCF on the structure and activity of the trypsin were carried out using electronic absorption and fluorescence spectroscopy (type of quenching, binding constant, number of binding locations, thermodynamic parameters, synchronous fluorescence, FRET analysis, 2D, and 3D fluorescence analysis, effect of coexistent drugs and metal ions), FTIR, thermal stability, kinetic and molecular docking techniques. Fluorescence quenching and electronic absorption results showed that the quenching process was a static mode. The bonding process's main driving force was hydrogen bonding and van der Waals forces with negative enthalpy and entropy changes. Synchronous, 2D and 3D fluorescence analyses suggested that the binding of the FCF to trypsin leads to some microenvironmental and conformational changes in the enzyme. The thermal stability study indicated that the FCF and the trypsin interaction could lead to a higher T m point and stability for the enzyme. Additionally, kinetic studies showed that the FCF inhibited the trypsin activity in a mixed inhibition model. Molecular docking studies validated the above experimental results. Molecular docking simulations were conducted to assess the interactions between the FCF and the amino acid residues of trypsin. The findings demonstrate strong binding affinity scores within the active site of trypsin, which agree with fluorescence quenching, thermodynamic analyses, and solvent-accessible surface area (SASA) calculations. This consistency suggests that docking can effectively validate experimental observations, providing complementary insights into the molecular interactions governing the FCF binding and its potential impact on enzyme activity.