Photodisruption increases the free-radical reactivity of melanosomes isolated from retinal pigment epithelium

Melanin in vivo is usually packaged in melanosomes with protein coats that restrict direct interaction of the melanin with the surrounding medium. We found that disruption of the melanosomes by exposure to a pulsed laser increased the ability of the melanin radicals to oxidize NADPH in a photochemical reaction. Retinal pigment epithelial (RPE) melanosomes were prepared from fresh bovine eyes in 0.25 M sucrose. A reaction mixture of 7 mM NADPH, approximately 7500 RPE melanosomes, and 80 mM Tris buffer, pH 7.2, was prepared in a volume of 60 μl. Of the two 25-μl aliquots taken from this mixture, one was pre-exposed to the 2nd-harmonic output of a Q-switched Nd:YAG laser (532 nm, 1800 10-nsec pulses at 10 Hz), and then was exposed to an Argon ion continuous wave (CW) laser (488.1 and 514.5 nm) for five minutes. The other aliquot was exposed only to the Argon laser. The CW exposure excited the melanin radicals to a reactive state that oxidized NADPH, as assayed by the loss of absorbance at 340 nm. Native melanosomes oxidized less NADPH during Ar ⁺ laser pumping than did melanosomes pre-exposed to the YAG laser. The YAG laser's stimulatory effect on melanosomes reactivity increased as the total energy it delivered rose above 3.5 J (0.14 J/cm ²/pulse × 1800 pulses), up to a maximum NADPH oxidation at about 20 J (0.2 J/cm ²/pulse × 1800 pulses, beam broadened at higher pulse energy). Electron microscopic analysis of the melanosomes confirmed the progressive physical disruption of melanosomes as the YAG pulse energy increased.