PHOTOSENSITIZATION OF BOVINE SERUM ALBUMIN BY PTERIN: A MECHANISTIC STUDY

ANDRÉS H. THOMAS; CAROLINA LORENTE; PAULINA RUIZ CARMONA; M. LAURA DÁNTOLA

Foz do Iguaçu

Congreso; 12th Latin American Conference on Physical Organic Chemistry; 2013

12th Latin American Conference on Physical Organic Chemistry

Solar radiation induces modifications to different biomolecules and is implicated in the generation of human skin cancers. In particular, UV-A radiation (320-400 nm) causes chemical changes in biomacromolecules through photosensitized reactions. This indirect action may take place through photosensitized mechanisms, which can involve the generation of radicals (type I mechanism), e.g., via electron transfer or hydrogen abstraction, and/ or the production of singlet oxygen (1O2) (type II mechanism).[1] Pterins are a family of heterocyclic compounds widespread in living systems. 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 proteins. The only studies on processes photosensitized by pterins that affect proteins is a very recent work in which, the inactivation of tyrosinase, enzyme that catalyzes the first step in the biosynthesis of the melanin (melanogenesis), photoinduced by Ptr has been demonstrated. [4] Vitiligo is a skin disease that causes lack of pigmentation due to inactivation of enzymes in the melanogenesis. It has been demonstrated that in the skin of these patients there is accumulation of pterin derivatives with high levels of hydrogen peroxide (H2O2). [5] Therefore, the photochemistry and the photosensitizing properties of pterins are of particular interest for the study of this disease.Therefore, given the important biological and medical ramifications of the photosensitization of proteins by pterins, we set out to investigate the damage of bovine serum albumin (BSA) photoinduced by pterin (Ptr), the parent and unsubstituted compound of oxidized pterins. As a first step, before studying the damage to BSA photoinduced by Ptr, we have investigated the interaction between the two molecules and we have evaluated the association, determining the corresponding binding constant. Emission spectra and fluorescence lifetime of the acid form of Ptr (17 mM) were recorded at different temperature, in the presence of different concentrations of BSA (0 to 1 mM) at pH 6.0. The results obtained indicate that the quenching of singlet excited state of Ptr by BSA is mainly static, the value of the constant binding is (1.27 ± 0.03) 103 M-1 at 25 ºC. The low affinity of BSA for Ptr implies that, in most of the experiments carried out in the studies of photodamage, less than 1 % of the photosensitizer was bound to the protein. In order to evaluate the capability of Ptr to photoinduce damage to BSA, aqueous solutions of the protein were expose to UV-A (lexc = 350 nm) radiation in the presence of Ptr. The irradiated solutions were analyzed by UV/vis spectrophotometry, fluorescence spectroscopy, enzymatic determination of H2O2 and sodium dodecyl sulfate polyacrylamide gel electrophoresis. In this work, electrophoretic analysis revealed that the formation of compounds of molecular weights higher than that of BSA, suggesting that a cross-linking process takes place. Moreover, the fluorescence analysis of irradiated solutions showed a significant decrease in the intensity of tryptophan (Trp) fluorescence with de irradiation time, indicating that a part of the Trp residues are damage in the photosensitization process. The results showed that Ptr is able to photoinduce damage to BSA, and that the main mechanism involves an electron transfer-initiated process. [1] Cadet, J.; Sage, E.; Douki, T. Mutat. Res. 571, 3 (2005). [2] Ito K.; Kawanishi S. Biochem. 36, 1774 (1997). [3] Petroselli, G.; Dántola, M. L.; Cabrerizo, F. M.; Capparelli, A. L.; Lorente, C.; Oliveros, E.; Thomas A. H. J. Am. Chem. Soc. 130, 3001 (2008). [4] M. L. Dántola, A. D. Gojanovich, A. H. Thomas, Biochem. Biophys. Res. Commun., 424, 568 (2012). [5] Schallreuter, K. U.; Wood, J. M.; Pittelkow, M. R.; Gutlich, M.; Lemke, K. R.; Rodl, W.; Swanson, N. N.; Hitzemann, K.; Ziegler I. Science, 263, 1444 (1994).