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

doi:10.4236/ojpc.2013.34019

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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

Email: [email protected]

Received March 26, 2013; revised April 25, 2013; accepted May 1, 2013

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

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



are the fractions of NO and O2 melanin binding

sites bound by NO and O2 respectively, we have:

NO ONO

Vk k









 (1)

The Langmuir treatment yields [20]









NO NO 1NO

ad ad



 (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



 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

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

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



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)

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.

Open Access OJPC

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

-1

0.0 0.5 1.0 1.5 2.0 2.5

1 / V x 10

-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





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,

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-

Open Access OJPC

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*.









 

dSNAPdSNAP

SNAPexp

ttk t



 

 







(2a)

Substituting these values into the integrated equation

(see 20) subsequently afforded the value of





 

–4

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.





SNAPNO NO

 (1a)