The Role of Eumelanin in Generating Reactive Oxygen and Reactive Nitrogen in Solution: Possible Relevance to Keloid Formation

Julian M. Menter, Comnuan Nokkaew, Danita Eatman, Aquilla Sprewell, Natalia Silvestrov, Abrienne M. Patta, Sandra Harris-Hooker

Analytical Laboratory, Department of Pharmacology and Toxicology, Morehouse School of Medicine, Atlanta, USA.
Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, Atlanta, USA.
Office of Sponsored Research, Department of Pathology, Morehouse School of Medicine, Atlanta, USA.

DOI: 10.4236/ojpc.2013.34019                

Abstract

Recently, nitric oxide (NO) has been implicated as an epigenetic factor in keloids, a scarring disease occurring primarily in dark skinned people who have relatively high amounts of pigment melanin. In this work, we tested whether a melanin-mediated redox reaction involving adsorbed NO and O₂ can couple NO oxidation with O₂ reduction to form reactive oxygen species (ROS) or reactive nitrogen species (RNS) in vitro at pH 7.4. We measured the formation of reactive species that oxidize dihydrorhodamine123 (DHR) to fluorescent rhodamine123 in the presence and absence of sepia melanin. In separate experiments, we monitored NO concentration with 4,5-diaminofluorescein (DAF) by measuring the highly fluorescent NO-adduct, DAF-2T. We attempted to detect peroxynitrite with 5 μM 3-methyl-1,2-cyclopentanedione (MCP), a selective scavenger of peroxynitrite (IC50 = 3.6 μM for ONOO^- vs. 63.8 μM and >> 100 μM for NO and O₂^- respectively). However, MCP itself oxidized DHR. We found that in the absence of NO, melanin itself oxidizes DHR, with no loss of DAF-fluorescence (i.e. no net consumption of NO). In the presence of NO, there was a ~ 57% loss of DAF fluorescence, indicating that NOx is formed at the expense of NO. The data provided good fit (r² = 0.94) to a Langmuir adsorption isotherm, with pseudo first order rate k' = 8.2 × 107) s^-1 and adsorption coefficient Kad = 4.04 M^-1. Both of these parameters are consistent with a facile chemisorption reaction between NO and O₂ on the melanin surface. Possible reactions are a) NO and O₂^- → ONOO^- and/or b) 2NO + O₂ → 2NO₂. The latter reaction is disfavored in solution but is significantly accelerated on the melanin surface via an entropy effect.

Keywords

Melanin; Nitric Oxide; NOx; Redox

Conflicts of Interest

The authors declare no conflicts of interest.

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