{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,11]],"date-time":"2026-10-11T02:18:43Z","timestamp":1791685123005,"version":"4.3.4"},"reference-count":26,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2021,6,17]],"date-time":"2021-06-17T00:00:00Z","timestamp":1623888000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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\u2013250 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.<\/jats:p>","DOI":"10.3390\/ijms22126512","type":"journal-article","created":{"date-parts":[[2021,6,17]],"date-time":"2021-06-17T11:20:26Z","timestamp":1623928826000},"page":"6512","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":52,"title":["Free Radicals and ROS Induce Protein Denaturation by UV Photostability Assay"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5596-9295","authenticated-orcid":false,"given":"Paolo","family":"Ruzza","sequence":"first","affiliation":[{"name":"Padova Unit, Institute of Biomolecular Chemistry of CNR (ICB-CNR), Via F. Marzolo, 1, 35030 Padova, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Claudia","family":"Honisch","sequence":"additional","affiliation":[{"name":"Padova Unit, Institute of Biomolecular Chemistry of CNR (ICB-CNR), Via F. Marzolo, 1, 35030 Padova, Italy"},{"name":"Department of Chemical Sciences, University of Padua, Via F. Marzolo, 1, 35030 Padova, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rohanah","family":"Hussain","sequence":"additional","affiliation":[{"name":"Diamond Light Source Ltd., Harwell Science and Innovation Campus, Didcot OX11 0DE, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4667-6423","authenticated-orcid":false,"given":"Giuliano","family":"Siligardi","sequence":"additional","affiliation":[{"name":"Diamond Light Source Ltd., Harwell Science and Innovation Campus, Didcot OX11 0DE, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,6,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1016\/j.ejpb.2015.10.016","article-title":"Forced degradation studies of biopharmaceuticals: Selection of stress conditions","volume":"98","author":"Tamizi","year":"2016","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1002\/prot.340070302","article-title":"Protein secondary structure and circular dichroism: A practical guide","volume":"7","author":"Johnson","year":"1990","journal-title":"Proteins"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1002\/9780470110522.ch2","article-title":"Circular dichroism and its empirical application to biopolymers","volume":"31","author":"Johnson","year":"1985","journal-title":"Methods Biochem. Anal."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1016\/0076-6879(95)46006-3","article-title":"Circular dichroism","volume":"246","author":"Woody","year":"1995","journal-title":"Methods Enzym."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"7799","DOI":"10.1002\/anie.201003877","article-title":"Circular dichroism of amino acids in the vacuum-ultraviolet region","volume":"49","author":"Meierhenrich","year":"2010","journal-title":"Angew. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"142","DOI":"10.1107\/S0909049503024142","article-title":"CD12: A new high-flux beamline for ultraviolet and vacuum-ultraviolet circular dichroism on the SRS, Daresbury","volume":"11","author":"Clarke","year":"2004","journal-title":"J. Synchrot. Radiat."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1107\/S0909049511038982","article-title":"Circular dichroism beamline B23 at the Diamond Light Source","volume":"19","author":"Hussain","year":"2012","journal-title":"J. Synchrot. Radiat."},{"key":"ref_8","unstructured":"Carreira, E.M., and Yamamoto, H. (2012). 8.23 Spectroscopic Analysis: Synchrotron Radiation Circular Dichroism. Comprehensive Chirality, Elsevier."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1016\/j.foodchem.2016.08.133","article-title":"Spectroscopy reveals that ethyl esters interact with proteins in wine","volume":"217","author":"Ruzza","year":"2017","journal-title":"Food Chem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"E149","DOI":"10.1002\/chir.20924","article-title":"Measuring Circular Dichroism in a Capillary Cell Using the B23 Synchrotron Radiation CD Beamline at Diamond Light Source","volume":"22","author":"Javorfi","year":"2010","journal-title":"Chirality"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1016\/j.ijpharm.2014.06.008","article-title":"The effect of palmitoylation on the conformation and physical stability of a model peptide hormone","volume":"472","author":"Longo","year":"2014","journal-title":"Int. J. Pharm."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1007\/s00726-009-0440-7","article-title":"Profiling of residue-level photo-oxidative damage in peptides","volume":"39","author":"Grosvenor","year":"2010","journal-title":"Amino Acids"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"517","DOI":"10.1107\/S0909049505006953","article-title":"VUV irradiation effects on proteins in high-flux synchrotron radiation circular dichroism spectroscopy","volume":"12","author":"Wien","year":"2005","journal-title":"J. Synchrot. Radiat."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"794","DOI":"10.1016\/j.bbrc.2012.07.154","article-title":"A zinc complex of heparan sulfate destabilises lysozyme and alters its conformation","volume":"425","author":"Hughes","year":"2012","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1007\/978-1-4939-2230-7_14","article-title":"CD spectroscopy: An essential tool for quality control of protein folding","volume":"1261","author":"Siligardi","year":"2015","journal-title":"Methods Mol. Biol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"04B310","DOI":"10.1116\/1.4972113","article-title":"Gold nanoparticles increases UV and thermal stability of human serum albumin","volume":"11","author":"Capomaccio","year":"2016","journal-title":"Biointerphases"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1021\/bi00504a006","article-title":"Estimation of globular protein secondary structure from circular dichroism","volume":"20","author":"Provencher","year":"1981","journal-title":"Biochemistry"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"663","DOI":"10.1016\/S0021-9258(18)44992-7","article-title":"Electrophoretic Patterns, Colloid Osmotic Pressure, And Viscosity Of Serum Denatured By Ultraviolet Radiation","volume":"146","author":"Davis","year":"1942","journal-title":"J. Biol. Chem."},{"key":"ref_19","unstructured":"Neves-Petersen, M., Petersen, S., and Prakash Gajula, G. (2012). UV Light Effects on Proteins: From Photochemistry to Nanomedicine. Molecular Photochemistry-Various Aspects, Intech Open."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"3514","DOI":"10.1021\/cr0682047","article-title":"Hydroxyl radical-mediated modification of proteins as probes for structural proteomics","volume":"107","author":"Xu","year":"2007","journal-title":"Chem. Rev."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/2192-2853-1-5","article-title":"Dynamic fluorescence imaging of the free radical products of X-ray absorption in live cells","volume":"1","author":"Rappole","year":"2012","journal-title":"Opt. Nanoscopy"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1016\/S1011-1344(02)00231-2","article-title":"UV-B-induced formation of reactive oxygen species and oxidative damage of the cyanobacterium Anabaena sp.: Protective effects of ascorbic acid and N-acetyl-L-cysteine","volume":"66","author":"He","year":"2002","journal-title":"J. Photochem. Photobiol. B Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"551","DOI":"10.5516\/NET.2008.40.7.551","article-title":"Better Understanding of the Biological Effects of Radiation by Microscopic Approaches","volume":"40","author":"Kim","year":"2008","journal-title":"Nucl. Eng. Technol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"306","DOI":"10.2307\/3574267","article-title":"Estimation of Life Times and Diffusion Distances of Radicals Involved in X-Ray-Induced DNA Strand Breaks or Killing of Mammalian Cells","volume":"64","author":"Roots","year":"1975","journal-title":"Radiat. Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"805","DOI":"10.1042\/BJ20151227","article-title":"Protein oxidation and peroxidation","volume":"473","author":"Davies","year":"2016","journal-title":"Biochem. J."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1107\/S1600577514028161","article-title":"CDApps: Integrated software for experimental planning and data processing at beamline B23, Diamond Light Source","volume":"22","author":"Hussain","year":"2015","journal-title":"J. Synchrot. Radiat."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/12\/6512\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:17:47Z","timestamp":1760163467000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/12\/6512"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,6,17]]},"references-count":26,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2021,6]]}},"alternative-id":["ijms22126512"],"URL":"https:\/\/doi.org\/10.3390\/ijms22126512","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,6,17]]}}}