Chemical changes in bovine serum albumin photoinduced by pterin

THOMAS, ANDRÉS H.; LORENTE, CAROLINA; ROMAN, ERNESTO A.; DANTOLA, MARIA LAURA

Carlos Paz

Congreso; 13th Latin American Conference on Physical Organic Chemistry; 2015

Universidad Nacional de Córdoba

Solar radiation induces modifications to different biomolecules and isimplicated in the generation of human skin cancers. In particular, UV-Aradiation (320-400 nm) causes chemical changes in biomacromolecules throughphotosensitized reactions. This indirect action may take place throughphotosensitized mechanisms, which can involve the generation of radicals (Type Imechanism), e.g., via electrontransfer or hydrogen abstraction, and/ or the production of singlet oxygen (1O2)(Type II mechanism).[1] Pterins are a family of heterocyclic compounds widespread in livingsystems. Currently, it is known that pterins are able to oxidize DNA [2]and nucleotides [3] through photosensitizing processes. However,much less is known about its action on interaction with proteins. Two recentstudies have suggested that degradation of proteins photosensitized by pterin (Ptr), the parent and unsubstituted compound ofoxidized pterins, involves a Type I mechanism.[4,5] Vitiligo is a skin disorder characterized by the acquired loss ofconstitutional pigmentation, manifesting as white macules and patches, and theaccumulation of reduced and oxidized pterins. In diseased skin cells micromolarconcentration of pterins have been determined and epidermal albumin oxidationtakes place. Taking into account that oxidized pterins are present in human skinunder pathological conditions in which the protection against UV radiationfails due to the lack of melanin and that albumin is one of the most abundantproteins present in skin, the main aim of this work is to investigate thechemical changes on bovine serum albumin (BSA) expose to UV?A in the presenceof pterin (Ptr). The experiments were carried out at concentrations of Ptr ofthe same order of magnitude found in human skin affected by vitiligo.[7]After UV-A excitation, aqueous solutions containing BSA and Ptrwere analyzed by UV/visiblespectrophotometry, fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) coupled withdynamic light scattering detection. The results indicated that thephotoinduced oxidation of BSA by Ptr is a purely dynamic process and results inthe oxidation of the protein in at least two different and specific sites:tryptophan (Trp) and tyrosine (Tyr) residues. The Trp degradation results in afast decrease of the fluorescence intensity, which is observed beforestructural changes in SDS-PAGE analysis. In addition, Tyr residues contributeto dimerization of the protein, since dimers of Tyr residues were detectedclearly by fluorescence. The SEC analysis indicated unequivocally that Ptr photoinducescross-linking of BSA, most likely due to dimerization between Tyr residues.  [1]J. Cadet, et. al. Mutat. Res. 571, 3 (2005).[2]K. Ito, S. Kawanishi. Biochem. 36,1774 (1997).[3]G. Petroselli, et. al. J. Am. Chem. Soc. 130, 3001 (2008).[4]A. H. Thomas, et. al. J. Photochem.Photobiol. B: Biology, 120, 52 (2013).[5] M. L. Dántola, et. al. Biochem. Biophys. Res. Commun, 424, 568 (2012).[6] H. Rokos, et.al. J. Raman Spectrosc., 35, 125 (2004).[7] K. U. Schallreuter., et. al.  J. Invest.Dermatol.,116, 167 (2001).