Photosensitization of human serum albumin by Pterin

LARA O. REID; ERNESTO A. ROMAN; ANDRÉS H. THOMAS; M. LAURA DÁNTOLA

Congreso; 25st Inter American Photochemical Society Conference; 2016

Solar radiation inducesmodifications to different biomolecules and is implicated in the generation ofhuman skin cancers. In particular, UV-A radiation (320-400 nm) causes chemicalchanges in biomacromolecules through photosensitized reactions. This indirectaction may take place through photosensitized mechanisms, which can involve thegeneration of radicals (Type I mechanism), e.g.,via electron transfer or hydrogen abstraction, and/ or the production ofsinglet oxygen (1O2) (Type II mechanism).[1] Pterins are a family of heterocycliccompounds widespread in living systems. Currently, it is known that pterins areable to oxidize DNA [2] and nucleotides [3] throughphotosensitizing processes. However, much less is known about its action oninteraction with proteins. Recent studies have suggested that degradation of proteinsphotosensitized by pterin (Ptr), theparent and unsubstituted compound of oxidized pterins, involves a Type Imechanism.[4,5,6] Vitiligo is a skin disordercharacterized by the acquired loss of constitutional pigmentation, manifestingas white macules and patches, and the accumulation of reduced and oxidizedpterins. In disease skin cells micromolar concentration of pterins have beendetermined and epidermal albumin oxidation takes place.[7] Taking into account that oxidizedpterins are present in human skin under pathological conditions in which theprotection against UV radiation fails due to the lack of melanin and thatalbumin is one of the most abundant proteins present in skin, the main aim ofthis work is to investigate the chemical changes on human serum albumin (HSA)expose to UV?A radiation in the presence of pterin (Ptr). The experiments werecarried out at concentrations of Ptr of the same order of magnitude found inhuman skin affected by vitiligo.[8] After UV-A excitation, aqueous solutions containing HSAand Ptr were analyzedby UV/visible spectrophotometry, fluorescence spectroscopy, sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusionchromatography (SEC) coupled with dynamic light scattering detection. The results indicated that HSA can be damaged by Ptr through aphotosensitized process. Mechanisticanalysis suggests that the photosensitized process is initiated by an electrontransfer from the protein to the triplet excited state of Ptr. Thephotodamage to HSA results in the oxidation of the protein in at least twodifferent and specific sites: tryptophan (Trp) and tyrosine (Tyr) residues. TheTrp degradation results in a fast decrease of the fluorescence intensity, whichis observed before structural changes in SDS-PAGE analysis. In addition, Tyrresidues contribute to dimerization of the protein, since dimers of Tyrresidues were detected clearly by fluorescence. The SEC analysis indicated unequivocallythat Ptr photoinduces cross-linking of HSA.  [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] A. H. Thomas, et. al. J. Photochem.Photobiol. B: Biology, 141, 262 (2014).[7] H. Rokos, et.al. J. Raman Spectrosc., 35, 125 (2004).[8] K. U. Schallreuter., et. al.  J. Invest. Dermatol., 116, 167 (2001).