Abstract
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.
Highlights
Biotherapeutics are becoming the mainstream of new medicinal agents, from which monoclonal antibodies and peptides are of great pharmaceutical interest
The heating hypothesis was assessed by comparing the effects of heating from 5 ◦ C to against the far-UV irradiation induced at 23 ◦ C by scanning 50 repeated consecutive
Irradiation (Figure S1, insert, blue line). These results indicated that the transformation of dihydrorhodamine 123 (DHR-123) in the fluorescent Rhodamine 123 (Rh-123) was due to the action of free radicals generated by the UV irradiation of the aqueous buffer solution and not by a photo-oxidation process
Summary
Biotherapeutics are becoming the mainstream of new medicinal agents, from which monoclonal antibodies and peptides are of great pharmaceutical interest. The development of biopharmaceuticals is often hampered by the reduced or lack of stability during ageing under a variety of environmental factors such as temperature, light, and oxidation that is manifested by a loss of ordered structure or protein misfolding mirrored by the loss or change of function. Circular dichroism (CD) spectroscopy is the ideal technique to characterize and monitor the folding of protein in solution as a function of environmental factors such as temperature, pH, solvent polarity, salts, detergents, lipids, and ligand interactions [2,3,4]. Unlike macromolecular crystallography (MX) and NMR where detailed structural models of the proteins at atomic resolution can be achieved, CD spectroscopy provides a fast method to analyze the conformational behavior in solution to confirm that different environmental conditions more appropriate for MX and NMR measurements do not affect the protein folding observed under physiological conditions. There are many techniques that can be used to determine binding interactions such as fluorescence, isothermal titration
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