Human Insulin Enhances Avobenzone Photostability while Retaining Its Biological Activity

Abstract

Human insulin was selected as a model polypeptide to investigate avobenzone photoprotection because of its structural stability, dermatological relevance, and role in diabetes therapy. Avobenzone, a widely used chemical ultraviolet A (UVA) filter, undergoes photodegradation upon UV exposure, limiting its long-term efficacy in sunscreen formulations. This study systematically examined the interaction between insulin and avobenzone to evaluate its effects on photostability, binding behavior, and insulin bioactivity. The UV spectroscopy-based photodegradation studies demonstrated that insulin significantly enhances avobenzone photostability under both sunlight and UV irradiation. The interaction was characterized using ESI-MS/MS, fluorescence spectroscopy, 19F NMR spectroscopy, and molecular dynamics simulations. Mass spectrometry confirmed the formation of a noncovalent insulin–avobenzone complex, with up to three avobenzone molecules binding per insulin molecule. Fluorescence quenching studies revealed a binding affinity of 52.1 μM, with predominantly dynamic quenching at low avobenzone concentrations and mixed dynamic/static quenching at higher concentrations. 19F NMR studies using 4-fluoroavobenzone showed selective broadening of the enol tautomer, whereas the keto form remained largely unaffected at a 10:1 insulin-to-ligand ratio, indicating altered tumbling rate upon complexation. MD simulations revealed hydrogen bonding between Glu4/Glu13 of insulin and the enol hydroxyl group of avobenzone and estimated the binding affinity order: enol-1 > enol-2 > keto tautomer. These interactions, along with anchoring within insulin cavities and restricted rotational mobility on the protein surface, are proposed to suppress photoketonization and enhance avobenzone photostability. Importantly, the insulin–avobenzone complex retained biological activity, indicating that insulin function is preserved upon complexation. These findings provide a proof of concept that insulin can improve avobenzone photostability without compromising biological activity, with potential relevance to diabetes-associated dermatological complications.