Time-Resolved Detection of Melanin Free Radicals Quenching Reactive Oxygen Species

Abstract

Melanin, a ubiquitous, heterogeneous biological polymer composed of many different monomers, contains a population of stationary, intrinsic semiquinone-like radicals. Additional extrinsic semiquinone-like radicals are reversibly photogenerated with visible or UV irradiation. The free radical chemistry of melanin is complex and not well characterized, especially the photochemistry of melanin in the presence of oxygen. To determine directly how melanin reacts in the presence of oxygen, time-resolved electron paramagnetic resonance (TREPR) spectroscopy was used to examine melanin free radical chemistry in human retinal pigment epithelium (RPE) cells under aerobic and anaerobic conditions. A TREPR difference spectrum was used to explore the nature of melanin chemistry in the presence of oxygen. The position and symmetrical line shape of the TREPR three-dimensional difference spectrum shows that when reactive oxygen species (ROS) are scavenged, only one of the two or more chemically different melanin free radical species participates in ROS scavenging. This protective melanin radical species exists in both the extrinsic and intrinsic populations of melanin free radicals, allowing melanin to protect the RPE from toxic species in both the light and dark.

Photoprotection of human retinal pigment epithelium cells against blue light-induced apoptosis by melanin free radicals from Sepia officinalis

“…Particularly, these additional extrinsic radicals likely correspond to the most emissive spike making the higher field (347.8-348.2 mT) contribution to the Sepia-RPE 3D spectrum. Similar asymmetrically increased TREPR-detected emission at higher field is common in previously reported spectra (14,48). However, the RPE spectra (Fig.…”

“…This work provides convincing evidence supporting a photoprotective role for melanin free radicals in vision, likely caused by the aggregated melanin free radical system serving as a versatile light and reactive species buffer in the RPE (14,48,53). That Sepia-RPE cultures thrived more than RPE cultures even in the dark despite increased intracellular melanin free radical concentrations strongly indicates that melanin free radicals generally promote RPE health.…”

“…Substantial evidence supports a phototoxic role of RPE melanin, especially in aged cells, including production of superoxide anions and hydroxyl radicals that are implicated in RPE cell death (39)(40)(41)(42)(43)(44)(45). However, it is also believed that RPE melanin serves a protective role in vision by absorbing light and scavenging free radicals and ROS (12,18,(46)(47)(48). We recently used TREPR to account for the ability of RPE melanin to serve as an efficient light absorber (14) and to directly detect melanin free radicals scavenging ROS in RPE cells (48).…”

“…However, it is also believed that RPE melanin serves a protective role in vision by absorbing light and scavenging free radicals and ROS (12,18,(46)(47)(48). We recently used TREPR to account for the ability of RPE melanin to serve as an efficient light absorber (14) and to directly detect melanin free radicals scavenging ROS in RPE cells (48).…”

“…For the RPE cells, the consequence of the Sepia melanin fast rise with increasing laser power is that not only are there about six times as many intracellular, stationary intrinsic radicals present in Sepia-RPE cells compared with RPE cells, but also many more photogenerated, mobile extrinsic radicals present at lower light intensities. These additional free radicals in the Sepia-RPE cells compared with the RPE cells are expected to result in more efficient light absorption (14) and ROS͞reactive species quenching (48).…”