Chemical Modifications of Globular Proteins Phototriggered by an Endogenous Photosensitizer
[EN] The main goal of the present work was to investigate the damages photoinduced by pterin (Ptr), an endogenous photosensitizer present in human skin under pathological conditions, on a globular protein such as ubiquitin (Ub). Particular attention has been paid on the formation of covalent adducts between Ptr and the protein that can behave as photoantigen and provoke an immune system response. Here, a multifaceted approach including UV-visible spectrophotometry, fluorescence spectroscopy, electrophoresis, size exclusion chromatography, and mass spectrometry is used to establish the Ub changes triggered by UV-A irradiation in the presence of Ptr. Under anaerobic conditions, the only reaction corresponds to the formation of a covalently bound Ptr-Ub adduct that retains the spectroscopic properties of the free photosensitizer. A more complex scheme is observed in air-equilibrated solutions with the occurrence of three different processes, that is, formation of a Ptr-Ub adduct, dimerization, and fragmentation of the protein. ; The present work was partially supported by Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET-Grants PIP 112-200901-0304 and PIP 11220120100072CO), Agencia de Promocion Cientifica y Tecnologica (ANPCyT-Grants PICT-2015-1988 and PICT 2016-00130), Universidad Nacional de La Plata (UNLP-Grant X712 and X840), and Universidad de Buenos Aires (UBA-Grant 000110BA). Funding from the Programa CSIC de Cooperacion Cientifica para el Desarrollo (iCOOPLight project ref 20105CD0017) and Spanish government (PGC2018-096684-B-I00) is gratefully acknowledged. The Ultraflex II (Bruker) TOF/TOF mass spectrometer was supported by a grant from ANPCYT, PME2003 No. 125, and the ESI-MS Q Exactive, Thermo Scientific, by a grant from ANPCYT, PME2011-PPL2-0009, CEQUIBIEM, DQB, FCEN, UBA. The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia. This proteomics laboratory is a member of Proteored, PRB3 and is supported by grant PT17/0019, of the PE I+D+i ...
