Ascorbate Enhances Photogeneration of Hydrogen Peroxide Mediated by the Iris Melanin[dagger]

Photochemistry and Photobiology, May/Jun 2008 by Wielgus, Albert R, Sarna, Tadeusz

EPR spectroscopy. UV- and light-induced changes in the concentration of melanin radical centers and ascorbyl radicals were monitored using EPR spectroscopy. The measurements were carried out using a Bruker ESP 300 E spectrometer operating at X-band (9.5 GHz) and equipped with 100 kHz field modulation. EPR spectra were recorded in the normal magnetic field sweeping mode or the kinetic mode with the magnetic field (B) fixed at the value B corresponding to maximal signal intensity and the time changes of signal amplitude were monitored. Melanin sample (200 �L) containing AscH- was placed in a flat quartz cell (pathlength 0.3 mm) designed for EPR measurements. The sample was irradiated directly in situ in the resonant cavity, which was being blown with nitrogen to prevent sample overheating. Before every measurement, the EPR cell was washed in alkaline solution of 30% H^sub 2^O^sub 2^ to remove all remains of the previous melanin sample. Then, it was repeatedly flushed with water, methanol and was dried with an air stream.

Ascorbate photo-oxidation. Ascorbate oxidation during irradiation of melanin samples was monitored spectrophotometrically at 265 nm (35) using the equipment described above. Changes in ascorbate absorbante accompanying oxygen photoconsumption were recorded directly in the EPR quartz flat cell. During measurements of H^sub 2^O^sub 2^ photoproduction, small atiquots of irradiated melanin sample were removed, at selected time intervals, from the photoreactor, centrifuged at 16 800 g for 2 min, and the ascorbate absorbance in the supernatant was recorded.

Statistical analysis. Standard errors of at least three measurements were calculated with a level of confidence of ~95%.

RESULTS

Photogeneration of hydrogen peroxide and photo-oxidation of exogenous ascorbate

Irradiation of bovine irides or their homogenates with UV-VIS light (296-680 nm) resulted in a formation of H^sub 2^O^sub 2^ (Table 1, Fig. 1a). The rate of photoinduced accumulation of H^sub 2^O^sub 2^ and its highest concentration in irradiated samples depended on the presence of ascorbate (Table 1, Fig. 1a). Thus, 50 �M ascorbate increased the rate of accumulation of H^sub 2^O^sub 2^ and its maximal concentration up to three times. Although H^sub 2^O^sub 2^ level detected after irradiation of bovine irides was lower than after irradiation of bovine iris homogenate, preincubation of the iris in 1 mM sodium azide resulted in virtually identical accumulation of H^sub 2^O^sub 2^ in both samples (Table 1, Fig. 1a). Similar effect was also observed in samples containing exogenous ascorbate. Highest accumulation of photoinduced H^sub 2^O^sub 2^, further enhanced by exogenous ascorbate, was observed in samples of homogenates of human iris (Table 1). In such samples, SOD had only a marginal effect on both the rates and maximal level of H^sub 2^O^sub 2^ accumulation (Fig. 1b). As expected, photoformation of H^sub 2^O^sub 2^ in samples with exogenous ascorbate was accompanied by its photo-oxidation (Fig. 1c). Although the initial concentration of ascorbate in samples shown in Fig. 1b and c was different, the kinetics of H^sub 2^O^sub 2^ accumulation in Fig. 1b and of ascorbate depletion in Fig. 1b is surprisingly similar, suggesting that both processes are interrelated. The key role of the iridial melanin in photoinduced generation of H^sub 2^O^sub 2^ and the accompanying photo-oxidation of ascorbate can be inferred from the results obtained on samples of iris homogenates and purified melanosomes isolated from bovine irides (Fig. 2). Both photoprocesses exhibit significant wavelength dependence-for melanosomes isolated from bovine irides the rates of photoproduction of H^sub 2^O^sub 2^ and of photo-oxidation of ascorbate at 312 nm are about 18-fold higher than at 480 nm. Similar spectral dependence of the photoprocesses is observed for homogenates of bovine irides: the ratio of rates of photogeneration of H^sub 2^O^sub 2^ at wavelengths 312 nm and 489 nm is 16 and that of photo-oxidation of ascorbate is about 15. Significantly, irradiation of ascorbate solution in PBS, without melanosomes or iris homogenate, with UV-VIS light, resulted in generation of only trace amounts of H^sub 2^O^sub 2^ (Table 1). After normalizing to equal melanin concentration, it is apparent that homogenates of human irides induced the fastest photo-oxidation of ascorbate (Fig. 3a). The initial rate of AscH- photo-oxidation in homogenates of human blue irides was ~3 and ~10 times higher than in samples containing bovine irides and ascorbate solution in PBS, respectively. Interestingly, a slow but noticeable oxidation of ascorbate in homogenates of human irides was also observed in the dark. An action spectrum for the photo-oxidation of ascorbate mediated by homogenate of human irides is shown in Fig. 3b. In spite of substantial standard deviation of the experimental data points, spectral dependences of the photoinduced oxidation of ascorbate, mediated by purified melanosomes, and by homogenates of bovine and human irides, exhibit significant similarities (Fig. 2b and 3b); i.e. the rate of ascorbate photo-oxidation decreases strongly with increasing wavelength, being at 664 nm almost 200-fold slower than that at 312 nm.