Open Journal of Physical Chemistry, 2013, 3, 157-162
Published Online November 2013 (http://www.scirp.org/journal/ojpc)
http://dx.doi.org/10.4236/ojpc.2013.34019
Open Access OJPC
The Role of Eumelanin in Generating Reactive
Oxygen and Reactive Nitrogen in Solution:
Possible Relevance to Keloid Formation
Julian M. Menter1, Comnuan Nokkaew2, Danita Eatman3, Aquilla Sprewell1, Natalia Silvestrov3,
Abrienne M. Patta1, Sandra Harris-Hooker2
1Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, Atlanta, USA
2Office of Sponsored Research, Department of Pathology, Morehouse School of Medicine, Atlanta, USA
3Analytical Laboratory, Department of Pharmacology and Toxicology, Morehouse School of Medicine, Atlanta, USA
Received March 26, 2013; revised April 25, 2013; accepted May 1, 2013
Copyright © 2013 Julian M. Menter et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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 mela-
nin-mediated redox reaction involving adsorbed NO and O2 can couple NO oxidation with O2 reduction to form reac-
tive 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-cyclopenta-
nedione (MCP), a selective scavenger of peroxynitrite (IC50 = 3.6 µM for ONOO vs. 63.8 µM and >> 100 µM for NO
and respectively). However, MCP itself oxidized DHR. We found that in the absence of NO, melanin itself oxi-
dizes 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 mela-
nin surface. Possible reactions are a) NO and
2
O
2
O

 ONOO and/or b) 2NO + O2  2NO2. The latter reaction is dis-
favored in solution but is significantly accelerated on the melanin surface via an entropy effect.
Keywords: Melanin; Nitric Oxide; NOx; Redox
1. Introduction
Pigment eumelanin binds a wide variety of compounds
[1] and can act as a redox reagent [2,3]. Depending on its
chemical environment, melanin can either donate or
accept electrons from ambient molecules and/or couple
the oxidation of one compound with the reduction of
another [4-6]. Melanin is well known to undergo auto-
xidation as well as Fenton chemistry with bound or
adventitious transition metals (usually iron or copper) to
produce .OH radicals. Depending on the relative metal
and pigment concentrations, melanin may act as either an
antioxidant or a pro-oxidant [7,8].
People of color are particularly susceptible to keloids,
a recalcitrant consequence of aberrant wound healing,
characterized by excessive collagen deposition that ex-
tends beyond the original wound [9-11]. The etiology of
keloids is complex, and both genetic [12] and epigenetic
[13] factors are involved. Recent work [14-16] has indi-
cated that nitric oxide (NO) enhances the synthesis of
type I collagen by dermal fibroblasts and that the keloid
lesion may arise from the NO/cGMP signaling path-
way. The particular susceptibility of darkly-pigmented
persons to keloids is intriguing, and it leads one to ask
whether pigment itself may play an active role in keloid
pathogenesis, given its ability to bind and mediate redox

J. M. MENTER ET AL.
158
reactions of bioactive molecules.
Recently, [4] we demonstrated that sepia melanin sca-
venged NO in solution through a cellulose dialysis mem-
brane. Crippa et al. [17] demonstrated that molecular O2
was adsorbed by melanin, and our observation of NO sca-
venging by melanin suggested a similar adsorption proc-
ess for NO. We thought it quite conceivable that the scav-
enged nitric oxide could be oxidized to additional reac-
tive nitrogen species (RNS; NOx) by direct redox reac-
tion with melanin and/or with adsorbed O2 or reactive
oxygen species (ROS) that arose in the course of melanin
autoxidation [2,3]. We used dihydrorhodamine 123 (DHR)
oxidation to measure the formation of ROS and RNS in
vitro at pH 7.4 in the presence and absence of added NO.
We show that melanin can adsorb NO and couple the
oxidation of NO with the reduction of O2.
2. Methods
Buffer: All experiments were carried out in 0.1 M phos-
phate buffer (“buffer”) pH 7.4 made up in ultrapure de-
ionized water. In several experiments, buffer was passed
through a Chelex-100 resin column (8.0 × 1.0 cm) be-
fore use. This treatment had no effect on the results.
Melanin preparation: Impure sepia melanin was ob-
tained as a kind gift from Dr. Miles Chedekel (Melan-
Ink@). Protein was removed by incubating this sample at
60˚C for 30 min in 0.1 M HCl followed by predialysis
through a Spectropore membrane (MW cutoff 6 - 8 kD)
into 100 mL 0.1 M phosphate buffer, pH 7.4/0.1 M
EDTA, followed by two changes of 0.1 M buffer alone.
Nitric oxide-generating compounds: We used fresh-
ly-prepared stock solutions of 0.1 mg/ml S-nitorso-N-
acetyl-DL-penicillamine (SNAP; Sigma Chemical Co.)
dissolved in 2.0 ml 95% ethanol and diluted to 10 ml
with buffer (final concentration 4.5 × 10−5 M). Donors
(250 µl of 0.02 mg/ml diluted stock) were added to test
and control dialyzates in a total of 22 ml in a 25-ml
graduated cylinder under mechanical stirring. All reac-
tions involving use of SNAP were carried out in subdued
light.
Detection of nitric oxide: Nitric oxide reacts with 4,5-
diaminofluorescein (DAF) in the presence of molecular
O2 to form a highly fluorescent triazole (DAF-2T) [18].
Fluorometric measurements were made on 200 µl ali-
quots of control and test dialyzates, which were diluted
to 1.0 ml in separate plastic cuvettes. At time t = 0, 1.0
ml of SNAP solution was added to each cuvette and the
DAF-2T fluorescence intensity (excitation 495 nm, emis-
sion 515 nm) measured in a Perkin-Elmer 650 - 40 Fluo-
rescence Spectrophotometer (Perkin Instruments, Nor-
walk, CT) as a function of time. The dialyzates of the
control and test samples (200 µl) were analyzed for NO
at times ranging from 0 to 120 minutes. Steady-state
fluorescence levels were reached at 60 minutes under our
experimental conditions. Data were expressed as the ratio
sample/control (SNAP alone) at t = 100 minutes.
The Interaction of Sepia Melanin with nitric oxide:
The reaction system consisted of 22 ml 0.1 M phosphate
buffer (pH = 7.4) in a 25 ml graduated cylinder, sur-
rounding dialysis bags that contained either a 5 ml sus-
pension of 5 mg sepia melanin or 5 ml buffer alone (see
below). Nitric oxide levels were measured from the test
and control dialysis filtrates as previously described [4].
Detection of NOx: Oxidation of dihydrorhodamine
123 (DHR): Dialysis systems were prepared as before,
with the addition of 1.0 ml of 2.0 mM dihydrorhodamine
123 (DHR; Life Sciences) in 25 ml of total solution.
Control systems were a) SNAP (NO) alone (blank); b)
SNAP (NO) + DHR (NO control); c) Melanin + DHR
(melanin control). Test systems contained d) Melanin +
DHR + SNAP (NO). (Oxidized) rhodamine fluorescence
(excitation 475 nm; emission 540) was recorded for 0.5
ml aliquots of test and control systems as functions of
time.
Estimation of the rate of NO oxidation: After cor-
recting the DHR oxidation curves for melanin-produc-
ed .OH, which oxidizes DHR [2,3,19], we used the initial
part of the DHR oxidation curves which yielded rea-
sonably straight lines and avoided build-up of secondary
RNS products (e.g. NO2) that could oxidize DHR [19].
Under these conditions, the rate of DHR oxidation is
taken to be equal to the rate of disappearance of NO. For
data analysis, the net oxidation of DHR by NOx was de-
termined by subtracting the contribution of each control
curve (see Figure 1, below). The corrected rate of DHR
oxidation by NO is proportional to the melanin-mediated
formation of NOx. The steady state concentration of NO
(t) is proportional to added SNAP concentration at t = 0.
We found the relationship between Rhodamine fluores-
cence and NOx concentration by means of a standard
curve.*
*For further discussion, see Appendix.
Langmuir adsorption isotherm of melanin-coupled
redox reaction(s) [4,20]: The Langmuir adsorption iso-
therm assumes that the reaction takes place with the re-
actants chemisorbed to the melanin surface. If θNO and
2
O

are the fractions of NO and O2 melanin binding
sites bound by NO and O2 respectively, we have:
2
NO ONO
Vk k




 (1)
The Langmuir treatment yields [20]




NO NO 1NO ad ad
KK   (2)
where [NO] is the solution concentration of NO in M, Kad
= ka/kd is the adsorption coefficient, i.e. the ratio of ad-
Open Access OJPC

J. M. MENTER ET AL. 159
Figure 1. Oxidation of DHR by NO (white circles), Eumela-
nin (black triangles) and Eumelanin/NO system (white tri-
angles) in 0.1 M phosphate buffer, pH 7.4 (see text). DHR is
slowly autoxidized (black circles). In the absence of added
melanin, NO slowly oxidizes DHR (white circles). Melanin
itself oxidizes DHR at a significantly greater rate with sig-
nificant upward concavity (black triangles); the additional
increases in DHR oxidation and concavity in the presence of
NO suggests the generation of NOx (white triangles).
sorption/dissociation constants of NO on the melanin
backbone, having the units of M1. We assume that
2
O
kk

 in O2 saturated (aerated) solution, is equal to
the pseudo first order rate constant for the bimolecular
reaction between O2 and NO on the melanin surface
having the dimensions of s1 (see [17,20] for discussion).
Substitution of (2) into (1) followed by inversion
yields


11 NO1
ad
VkK k

  (3)
Thus, if Langmuir mechanisms are operative, slopes of
1/V vs. 1/[NO] will yield a straight line with slope
1/k'Kad and intercept 1/k' from which parameters k' and
Kad can be found.
The steady state concentration, [NO]ss was estimated
from rate constants for formation and decline of NO in
consecutive reactions. These constants were found from
the approximate lifetimes of SNAP (~5 hr,) and NO de-
composition in aqueous solution (~2 s). Under these con-
ditions, NO is consumed virtually as fast as it is formed,
and [NO] could be assumed to be invariant with time.
Using these values, the integrated rate equation [20] ul-
timately yielded the value:


*
– 4
0
ss
NO1.12 10SNAP

  .
*For further discussion, see Appendix.
Statistical analysis: We used Student’s t test for small
samples. Data were expressed as mean ± SD. Differences
were considered significant for p < 0.05.
3. Results
DHR oxidation: Figure 1 shows the results of a repre-
sentative experiment (n = 4) in aerated solution. Control
groups were DHR alone (black dots); DHR + NO in the
absence of melanin (white dots) and melanin + DHR
(black triangles). DHR alone was slowly autoxidized to
fluorescent rhodamine 123. Nitric oxide modestly in-
creased the rate of DHR oxidation Melanin efficiently
oxidized DHR with significant upward concavity. The
test system, melanin + NO + DHR (white triangles), fur-
ther increased the rate of growth and concavity. Qualita-
tively similar results were obtained for each experiment.
Attempts to Detect NOx; Consumption of NO in
solution: At 5 µM, 3-methyl-1,2-cyclopentanedione (MCP;
Aldrich Chemical Co.) selectively scavenges ONOO-
(IC50 = 3.6 µM for ONOO vs. 63.8 µM and >> 100 µM
for NO and 2
O

respectively [21]. Although we origi-
nally planned to use MCP to detect peroxynitrite, this did
not prove successful, since MCP itself oxidized DHR,
(not shown). However, we did test for NO consumption
in the test system and control samples by using 4,5-dia-
minofluorescein (DAF-2; Sigma Chemical Co.) which
forms a highly-fluorescent triazole adduct with NO, (DAF-
2T), [18] and which does not react with DHR. Figure 2
shows that 5 µM MCP scavenges NO at ~20% (Figure
2(b)). Nitric oxide consumption by melanin alone in the
course of DHR oxidation does not occur (compare Fig-
ures 2(b) with 2(c)). However, the simultaneous pres-
ence of melanin and NO (Figure 2(d)) resulted in con-
sumption of NO by an additional 57%, suggesting con-
comitant NOx formation at the expense of NO.
(a) (b) (c) (d)
1
RELATIVE DAF FL UORESC ENCE
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SNAP (NO)
SNAP + MCP(AV)
MEL + MCP (AV)
TEST (AV)
Figure 2. Disappearance of NO (SNAP) in the presence of
sepia melanin in oxygen-saturated solution. DAF forms a
highly fluorescent triazole DAF-2T in presence of NO. MCP
(5 µM) scavenges virtually all ONOO (IC 50 = 3.6 µM), but
only slightly scavenges NO (IC 50 = 63.3 µM), Taken to-
gether, Figures 1 and 2 suggest NOx is formed at the ex-
pense of NO. (a) SNAP (NO) alone; (b) SNAP + MCP; (c)
Melanin + MCP; (d) Melanin + MCP + SNAP. Data are
expressed as mean ± S.D, n = 4.
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J. M. MENTER ET AL.
160
Kinetic Analysis of Melanin-Mediated NOx Forma-
tion: Previously, we and others have found that melanin
can couple oxidation of one species with reduction of
another. To test the hypothesis that melanin can mediate
a redox reaction involving NO [4] and O2 [17] adsorbed
on its surface, we analyzed our data according to a sim-
ple Langmuir adsorption isotherm [20].
Figure 3 shows a Langmuir plot of 1/V vs. 1/[NO].
This plot yielded a straight line, in good agreement with
the Langmuir model (r2 = 0.94). From the slope and in-
tercept, we obtained a value of pseudo first order con-
stant k' = 8.2 × 107 s1 and adsorption coefficient Kad =
4.04 M1.
4. Discussion
In the absence of sepia melanin, NO in aerated solution
slowly oxidizes indicator DHR. Sepia melanin alone
oxidizes DHR rapidly, but the addition of NO still further
increases the rate of DHR oxidation. Figure 1 indicates
that there is a synergistic effect in DHR oxidation when
both melanin and NO are present.
Sepia melanin is known to consume O2 in aerated
solution, producing H2O2 via a superoxide intermediate
[2,3]. Neither superoxide H2O2 nor physiological NO
concentrations will oxidize DHR, however, OH does oxi-
dize DHR to fluorescent Rhodamine123 [19]. Hydroxyl
1 / [NO] x 10
9
M
-1
0.0 0.5 1.0 1.5 2.0 2.5
1 / V x 10
8
M
-1
min
0.1
0.2
0.3
0.4
0.5
1/NO(corr) x 10 9 vs 1/sl ope (av) x 10 -10
Plot 1 Regr
Figure 3. Langmuir Adsorption curve for NO + O2 coupled
redox reaction on sepia melanin surface. After correction
for production of ROS by melanin, oxidation of DHR by
NO in solution and spontaneous oxidation of DHR by am-
bient O2 (see Figure 1), rates of production of NOx were
measured by rhodamine 123 fluorescence measurements
(see methods section). Steady-state [NO] as a function of
time was estimated from SNAP decomposition rates and
from the lifetime of NO in aqueous solution (see text). Plots
of 1/V vs. 1/[NO] afforded a straight line, (r2 = 0.94) that
intersected the 1/V axis above the origin, strongly indicating
a chemisorption mechanism, with adsorption coefficient Kad
= 4.04 M−1 and k' = 4.98 × 109 min−1 = 8.2 × 107 s−1.
radical could be generated from decomposition ONOOH
at pH 7.4, [20] natural sepia melanin, [3] as well as ad-
ventitious and/or melanin-bound iron and copper ions.
Bound transition metals are very difficult to remove,
even after exhaustive dialysis against EDTA [1].
Figure 2 shows that under these experimental condi-
tions, the NOx scavenger MCP does not scavenge ROS
(compare Figures 2(b) and (c)). However, MCP will
oxidize DHR (not shown). As indicated in Figure 2, 5.0
µM MCP scavenges ~20% of NO. There is an additional
57% NO consumption in the presence of added NO, pre-
sumably via formation of NOx. The results from Figure
2 confirm that the DHR-oxidizing species from melanin
in the absence of NO is not NOx.
Crippa et al. [17] demonstrated that molecular O2 is
adsorbed by melanin. We have observed melanin sca-
venging of NO that suggests a similar adsorption process
[4]. The observed fit of our data to a simple Langmuir
adsorption isotherm strongly suggests that the melanin-
mediated reactions leading to increased DHR oxidation
do involve adsorbed NO and O2 species. It seems rea-
sonable that sepia melanin could couple NO oxidation
with oxygen reduction to 2. Superoxide and NO might
then combine to form ONOO– in a fast reaction
O


2
101 1
NO O1.6 10Msk  [22]. Alternatively, 2 ad-
sorbed NO molecules in close proximity to molecular O2
could react to form NO2 and other NOx species. In the
absence of melanin the reaction of 2 molecules of NO
with one molecule of O2 to afford 2 molecules of NO2 is
slow, (pseudo second order constant,
2
41
NO O410M sk1





) at NO saturation, (~2 mM in
aqueous solution), because it depends on a simultaneous
attack of two molecules of NO on a molecule of O2 [23].
However, in adsorbing (“ordering”) NO and O2 on its
surface, melanin might well facilitate formation of NO2,
by significantly decreasing the entropy of the reaction.
Our data does not presently allow distinction between
these mechanisms.
Although comparison of our rate constant with solu-
tion data is not straight-forward because of complications
having to do with the melanin surface, our value of 8.2 ×
107 s1 is in reasonable agreement with a value obtained
from Crippa [17] who used a more rigorous treatment to
obtain a value of 5.7 × 106 s1 for reduction of dioxygen
by conduction band electrons of colloidal melanin (for
discussion, See [17]).
Hsu et al. [16] found that keloid tissue expression
showed significant increases in iNOS gene, NOx produc-
tion, type I collagen mRNA, and type I collagen protein
expression compared to normal fibroblasts. Our present
work demonstrates that melanin adsorbs NO and medi-
ates oxidation to NOx, with possible epigenetic cones-
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J. M. MENTER ET AL. 161
quences. Although the consequences of these interactions
are not well known with respect to keloids, one might
gain insight by considering the closely-related case of
systemic sclerosis, where imbalances in NO metabolism,
type I collagen expression, and the occurrence of nitrated
proteins have been known to occur for certain disease
states [24-26]. Melanin sequestration of NO and its role
in generating toxic NOx might have similar effects on
normal fibroblasts that could possibly result in their
transformation to keloid fibroblasts. This latter question
is currently under investigation in our laboratory (Nok-
kaew et al., in progress).
5. Conclusions
a) Sepia melanin accelerates (couples) the oxidation of
NO to NOx and the reduction of molecular O2 in buffered
aqueous solution.
b) The reaction kinetics fit well to a simple Langmuir
adsorption isotherm, and the reaction therefore appears to
take place between adsorbed NO and O2 via a chemi-
sorption mechanism. We obtained a value of pseudo first
order constant 7
8.2 10 sk1

 and adsorption
coefficient Kad = 4.04 M1.
c) Such a reaction occurring in situ in dermal tissue
might have significant epigenetic consequences in keloid
formation or related pathologies.
6. Acknowledgements
This work was funded in part by GRANTS: MBRS
#GM08248, RCMI #8G12MD007602, and DOD # 911
NF-10-1 0448. There are no conflicts of interest.
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Appendix In this case, 21 , and NO is consumed as fast as it
is formed, leaving a steady-state NO concentration,
[NO]ss. This treatment yields the differential equation for
[NO]
kk
For analysis, of the raw data from the Langmuir plot, the
net oxidation of DHR by NOx was determined by
subtracting the contribution of each control curve (see
Figure 1). The rate of DHR oxidation is proportional to
the formation of NOx; the concentration of steady state
concentration of NO is proportional to added SNAP
concentration at t = 0. We found the relationship between
rhodamine 123 fluorescence and [NO] by means of a
standard curve made by known concentrations of
rhodamine B*.




 
2
11
dSNAPdSNAP
SNAPexp
ttk t
ko
  
 
 

kt
(2a)
Substituting these values into the integrated equation
(see 20) subsequently afforded the value of


 
–4
ss
N
O1.1210SNAP Mo

The steady state [NO] was estimated from the half-
lives of SNAP decomposition (τ ~ 5 hr) and NO decom-
position (τ ~ 2 s). These allowed estimation of the re-
spective rate constants for NO arising from decomposi-
tion of SNAP, (51
13.9 10sk


 and 1
20.347 sk


for decomposition of NO. Treating the process as two
consecutive reactions, of the form [20] we get:
*Since the absorption/emission maxima of rhodamine
B are red-shifted by ~1100 cm−1 with respect to rhoda-
mine 123 and the quantum yields of fluorescence, φf very
similar, we shifted the excitation/emission measurement
to 496 nm and 587 nm. The choice of excitation wave-
length was chosen so that the both samples absorbed the
same amount of excitation radiation.


12
x
SNAPNO NO
kk
 (1a)