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T. J. Sarna, G. Szewczyk, M. Sarewicz, M. Zareba, A. Kittell, T. Camenisch; Characterization of Metal-Ion-Binding and Free Radical Properties of Experimentally Photoaged Bovine and Porcine RPE Melanosomes. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2915.
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RPE melanin is involved in photoprotection of the human retina; however, its chronic exposure to intense visible light may modify melanin's antioxidant capacity and photoprotective efficiency. Here we characterized metal-ion-binding and free radical properties of in vitro photobleached RPE melanosomes from porcine and bovine eyes, our experimental model of photoaged human RPE melanosomes
Melanosomes, isolated from bovine and porcine RPE, were photobleached with intense blue light. Progress of melanosomes photobleaching was monitored by X-band electron spin resonance (ESR) and UV-VIS absorption spectroscopy. The effect of photobleaching of RPE melanosomes on their paramagnetic and metal-ion-binding properties was determined by high frequency ESR spectroscopy (W-band ESR) and saturation recovery ESR using paramagnetic Dy(III) as a unique molecular probe with a very rapid spin-lattice relaxation.
Although at X-band, ESR spectra of untreated and photobleached melanosomes were practically identical, except signal intensity, which gradually decreased with the degree of photobleaching, parameters of ESR spectra of photobleached melanosomes at W-band exhibited small but distinct differences that correlated with the extend of photobleaching. Saturation recovery ESR revealed that spin-lattice relaxation time of melanin free radicals was reduced by bound to melanin dysprosium ions. Again, the effect was more pronounced for photobleached pigment granules.
We have demonstrated that experimental photobleaching of bovine and porcine RPE melanosomes is accompanied by characteristic changes in their paramagnetic properties readily detectable by W-band ESR. Structural changes of melanosomes induced by photobleaching could also be monitored by saturation recovery ESR that indicated a substantially modified topography of the metal-ion-binding sites relative to melanin free radical centers. These changes could be responsible for the observed modification of antioxidant properties of experimentally photoaged melanosomes.
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