Human Insulin Enhances Avobenzone Photostability while Retaining Its Biological Activity

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.