Free Radicals and ROS Induce Protein Denaturation by UV Photostability Assay.
Oxidative stress, photo-oxidation, and photosensitizers are activated by UV irradiation and are affecting the photo-stability of proteins. Understanding the mechanisms that govern protein photo-stability is essential for its control enabling enhancement or reduction. Currently, two major mechanisms for protein denaturation induced by UV irradiation are available: one generated by the local heating of water molecules bound to the proteins and the other by the formation of reactive free radicals. To discriminate which is the likely or dominant mechanism we have studied the effects of thermal and UV denaturation of aqueous protein solutions with and without DHR-123 as fluorogenic probe using circular dichroism (CD), synchrotron radiation circular dichroism (SRCD), and fluorescence spectroscopies. The results indicated that the mechanism of protein denaturation induced by VUV and far-UV irradiation were mediated by the formation of reactive free radicals (FR) and reactive oxygen species (ROS). The development at Diamond B23 beamline for SRCD of a novel protein UV photo-stability assay based on consecutive repeated CD measurements in the far-UV (180-250 nm) region has been successfully used to assess and characterize the photo-stability of protein formulations and ligand binding interactions, in particular for ligand molecules devoid of significant UV absorption.
['Ruzza|Paolo|P|0000-0002-5596-9295', 'Honisch|Claudia|C|', 'Hussain|Rohanah|R|', 'Siligardi|Giuliano|G|0000-0002-4667-6423']
D002942:Circular Dichroism D005609:Free Radicals D006361:Heating D011489:Protein Denaturation D011506:Proteins D017382:Reactive Oxygen Species D013057:Spectrum Analysis D014466:Ultraviolet Rays D014867:Water
protein photo-stability synchrotron radiation circular dichroism VUV and far-UV irradiation reactive oxygen species UV protein denaturation assay