Abstract
Purpose: :
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
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: retinal pigment epithelium • aging • oxidation/oxidative or free radical damage