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 t...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.展开更多
文摘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.
