Inactivation of tyrosinase photoinduced by pterin

M. LAURA DÁNTOLA; ALDANA D. GOJANOVICH; ANDRÉS H. THOMAS

Cordoba

Congreso; XI Encuentro Latinoamericano de Fotoquímica y Fotobiología (XI ELAFOT); 2012

Universidad Nacional de Río Cuarto

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) can induce damage to DNA and other macromolecules through photosensitized reactions [1]. This indirect action may be mediated by endogenous or exogenous photosensitizers and can take place through different mechanisms [2]. Although the photosensitized damage to DNA is well characterized, much less is known about proteins and very little has been studied on inactivation of enzymes caused by photosensitized processes. Pterins are a family of heterocyclic compounds widespread in living systems. Currently, it is known that pterins are able to oxidize DNA [3] and nucleotides [4] through photosensitizing processes. However, its action on proteins has not been studied yet. Vitiligo is a skin disease that causes lack of pigmentation due to inactivation of enzymes in the biosynthesis of melanin (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]. Tyrosinase is a copper-containing glycoprotein that in mammals catalyzes the first and rate-limiting step in melanin biosynthesis. Although it is recognized that pterins may be involved in the pathophysiology of vitiligo, no studies on the ability of these molecules to photoinduce the inactivation of enzymes of melanogenesis have been published. To investigate this, aqueous solutions containing the enzyme and the photosensitizer (pterin (Ptr)) were exposed to UV-A (lexc = 350 nm) for different period of time (pH = 6.5, 25 °C). After irradiation under different experimental conditions, the enzyme activity was determined. The method used consisted in monitoring the increase in absorbance at 475 nm, corresponding to the formation of L-Dopacroma as a function of reaction time. The enzyme activity was calculated from the slope of the absorbance vs. time curve [6]. The obtained results indicated that Ptr can fotoinactivate the tyrosinase, suggesting that the reaction starts with an electron transfer from the enzyme to the triplet excited state of the photosensitizer (3Ptr*), thus generating the radical cation of the enzyme (Tyr?+) and radical anion of the photosensitizer (Ptr?−). Tyr?+ can undergo oxidation which lead to the irreversible inactivation of the enzyme. Ptr?− can reduce the dissolved oxygen (O2) to generate superoxide anion (O2?-). In light of the overall data obtained, the following scheme was proposed to explain the mechanism of photoinactivation of tyrosinase by pterin.   References:         [1]. Cadet, J.; Douki, T. J. Invest. Dermatol. 131, 1005, 2011. [2]. Cadet, J.; Sage, E.; Douki, T. Mutat. Res. 571, 3, 2005. [3]. Ito K.; Kawanishi S. Biochem. 36, 1774,1997. [4]. 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. [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. [6]. Pomerantz, S. H.; Li, J. P. C. Method Enzimol. 17, 620. 1970.