A comparative study on the interaction of gold nanoparticles with trypsin and pepsin: thermodynamic perspectives

Abstract

Gold nanoparticles (AuNPs), as a type of biocompatible nanomaterial, possess many characteristics and show great potential in the biomedical field. Trypsin and pepsin are the main proteolytic enzymes in the digestive system. A comprehensive elucidation of the interaction between AuNPs and trypsin/pepsin is essential, mainly because AuNPs are likely to interact with trypsin and pepsin when they enter the organism through the digestive system. In this study, AuNPs with different particle sizes were prepared by reduction of HAuCl₄ with sodium citrate. The synthesized AuNPs were characterized using UV-vis spectroscopy, TEM and DLS. The interaction of 17 nm and 30 nm AuNPs with trypsin and pepsin was comprehensively investigated from the perspective of thermodynamics. AuNPs can act as a quencher to quench the fluorescence intensity of trypsin or pepsin, and the quenching mechanism mainly comes from static quenching, which is further confirmed by UV-vis analysis. The binding constant K_a and the Hill coefficient n of the interaction between AuNPs and trypsin/pepsin were calculated. The results show that AuNPs have a high affinity with trypsin or pepsin. The binding ability of trypsin to AuNPs is stronger than that of pepsin, and the binding ability of 17 nm AuNPs is stronger than that of 30 nm AuNPs. In addition, the positive cooperative effect of 17 nm AuNPs is more significant than that of 30 nm AuNPs. By evaluating the thermodynamic parameters, it is found that the interaction between AuNPs and trypsin/pepsin is a spontaneous, endothermic and entropy-driven process, and the main non-covalent force is the hydrophobic interaction. For the AuNP (17 nm)–trypsin system and the AuNP (30 nm)–pepsin system, the hydrophobicity of the Trp microenvironment of the two proteases decreases due to the interaction of AuNPs. AuNPs have different effects on the secondary structure of trypsin and pepsin, but AuNPs have more obvious effects on the secondary structure of pepsin than trypsin. The activity of trypsin/pepsin decreases with the addition of AuNPs.