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INTERNATIONAL FEDERATION OF PIGMENT CELL SOCIETIES · SOCIETY FOR MELANOMA RESEARCH

PIGMENT CELL & MELANOMA

Research
Melanins and melanogenesis: methods,
standards, protocols
Marco d’Ischia, Kazumasa Wakamatsu, Alessandra
Napolitano, Stefania Briganti, Jose-Carlos Garcia-Borron,
Daniela Kovacs, Paul Meredith, Alessandro Pezzella, Mauro
Picardo, Tadeusz Sarna, John D. Simon and Shosuke Ito

DOI: 10.1111/pcmr.12121
Volume 26, Issue 5, Pages 616–633

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Pigment Cell Melanoma Res. 26; 616–633 REVIEW

Melanins and melanogenesis: methods, standards,

protocols
Marco d’Ischia1, Kazumasa Wakamatsu2, Alessandra Napolitano1, Stefania Briganti3,

-Carlos Garcia-Borron4, Daniela Kovacs3, Paul Meredith5, Alessandro Pezzella1, Mauro Picardo3,
Jose
Tadeusz Sarna6, John D. Simon7 and Shosuke Ito2

1 Department of Chemical Sciences, University of Naples Federico II, Naples, Italy 2 Department of Chemistry, KEYWORDS melanin analysis/melanin synthesis/
Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan 3 Cutaneous Physiopathology melanin isolation/melanin spectral characterization/
Laboratory, San Gallicano Dermatological Institute, Rome, Italy 4 Department of Biochemistry and Molecular melanogenesis enzymes/method standardization
Biology, School of Medicine, University of Murcia, Murcia, Spain 5 Center for Organic Photonics & Electronics,
School of Mathematics and Physics, University of Queensland, Brisbane, Qld, Australia 6 Department of PUBLICATION DATA Received 2 March 2013,
Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland revised and accepted for publication 17 May 2013
7 Department of Chemistry, University of Virginia, Charlottesville, VA, USA published online 25 May 2013

CORRESPONDENCE Marco d’Ischia, e-mail: dischia@unina.it doi: 10.1111/pcmr.12121

Summary
Despite considerable advances in the past decade, melanin research still suffers from the lack of universally
accepted and shared nomenclature, methodologies, and structural models. This paper stems from the joint
efforts of chemists, biochemists, physicists, biologists, and physicians with recognized and consolidated
expertise in the field of melanins and melanogenesis, who critically reviewed and experimentally revisited
methods, standards, and protocols to provide for the first time a consensus set of recommended procedures to
be adopted and shared by researchers involved in pigment cell research. The aim of the paper was to define an
unprecedented frame of reference built on cutting-edge knowledge and state-of-the-art methodology, to enable
reliable comparison of results among laboratories and new progress in the field based on standardized methods
and shared information.

melanin research concerning (i) isolation and purification

Introduction
The melanins can be still regarded as the most enigmatic
pigments/biopolymers found in nature (Ito et al., 2011a).
Unlike the vast majority of natural pigments, the melanins
cannot be described in terms of a single well-defined
structure and, as a result, there still remains today a lack
of general consensus what actually melanin is. A variety
of definitions and models are found in the literature,
which reflect, however, an arbitrary use of terminology as
well as several assumptions and speculations that have
never been proven on experimental grounds. Crucial gaps
stem from the lack of standardized procedures and
methodologies, failure to take in due account melanin
properties and the consequences of harsh purification
procedures, a widespread tendency to compare materials
obtained under different conditions, to extrapolate data
referring to natural pigments from studies on synthetic
pigments, or to draw conclusions and implications from
observations made on unsuitable models.
The aim of this paper was to provide a critical
assessment of methodological and practical issues in
of natural melanins; (ii) preparation of synthetic model
pigments; (iii) physical, spectral, and chemical character-
ization; (iv) use of melanins and melanogenic enzymes for
biological studies; (v) preparation and assessment of
standard compounds for melanin research, including
commercially available pigments and enzymes.
Starting from a careful review of current methods and
standards, the paper provides a selection of guidelines
and procedures that have been verified and optimized,
when necessary, through an ad hoc experimental revi-
sion. Provided herein as integral part of the paper is also
the Appendix S1 section, which contains the first
systematic collection of reference data and experimental
protocols to be recommended as state-of-the-art for
future research in the field.
Definition and classification
The term ‘melanin’ was first coined by Berzelius in 1840
to refer to black animal pigments. Since then, it has been
widely used to indicate any black or dark brown organic

616 ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

pigment occurring throughout the phylogenetic scale
without any specific structural, biogenetic, or functional
implication. Nicolaus (1969) suggested a classification of
melanins into three main groups, eumelanins, pheomel-
anins, and allomelanins, the former two groups compris-
ing animal pigments and the latter encompassing the
broad variety of dark non-nitrogenous pigments of plant,
fungal, and bacterial origin. Pseudomonas and Aspergillus
fumigatus can produce in the presence of tyrosine a
eumelanin-like pigment termed pyomelanin via homogen-
tisic acid (Schmaler-Ripcke et al., 2009). Aspergillus
fumigatus can also synthesize melanin-type pigments
from 1,8-dihydroxynaphthalene, while Serratia marces-
cens (Trias et al., 1989) or the pathological fungus
Cryptococcus neoformans (Casadevall et al., 2000) can
produce similar pigments from alternate pathways. Plants
also produce dark phenolic pigments that have some-
times been referred to as catechol melanins, although the
term conveys no information about the broad diversity
and complexity of the polyphenolic precursors. To limit
confusion, Prota proposed a more restrictive usage of the
term ‘melanin’ to include only those pigments that are
formed intracellularly by the oxidation of tyrosine and
related metabolites (Prota, 1995), thus taking both the
biogenetic origin from tyrosine and the metabolic activity
of melanocytes (and, occasionally, related cell systems)
as stringent requisites.
Although in the authors’ opinion this restrictive classi-
fication of melanins remains valid, it is nonetheless
difficult to find alternative definitions for the broad variety
of dark phenolic pigments from plants and microorgan-
isms or for the diverse synthetic pigments of biomedical
and technological interest, which are driving much of
current progress in the field. Accordingly, it may be
convenient to maintain a wider, general purpose classifi-
cation, rooted in the tradition but revisited in light of recent
progress, that includes (i) sensu stricto melanins, (ii) the
dark phenolic pigments from lower organisms, and (iii)
synthetic pigments produced either chemically or enzy-
matically from natural precursors. The following set of
practical definitions is thus proposed and recommended.
Melanins: Pigments of diverse structure and origin
derived by the oxidation and polymerization of tyrosine
in animals or phenolic compounds in lower organisms.
Eumelanins: Black-to-brown insoluble subgroup of
melanin pigments derived at least in part from the
oxidative polymerization of L-dopa via 5,6-dihydroxyin-
dole intermediates.
Examples: sepia melanin, black hair melanin.
Pheomelanins: Yellow-to-reddish brown, alkali-solu-
ble, sulfur-containing subgroup of melanin pigments
derived from the oxidation of cysteinyldopa precursors
via benzothiazine and benzothiazole intermediates.
Examples: red hair melanin, hen feather melanin (it
should be noticed, however, that the red hair pigment is
rarely pure pheomelanin).
ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Methods in melanin research
Neuromelanins: Dark pigments produced within
neurons by the oxidation of dopamine and other
catecholamine precursors.
Example: substantia nigra melanin.
Pyomelanins: Dark pigments produced by microor-
ganisms mainly, but not exclusively, from homogen-
tisate.
Moreover, for all types of natural pigments, the term
‘melanin’ should be preceded by the natural source, for
example sepia melanin, hair melanin, while for synthetic
pigments the term ‘melanin’ (or eumelanin, pheomela-
nin whenever appropriate) should be preceded by that
of the precursor, for example dopamine melanin, 5,6-
dihydroxyindole melanin, cysteinyldopa-melanin, dihydr-
oxynaphthalene melanin.
Natural melanins
For the direct investigation of melanins, it is essential to
keep in due consideration certain peculiar and critical
properties of the pigments (Prota, 1992):
1. Melanins are not stable indefinitely. They may
undergo more or less profound structural degradation
on chemical treatment with acids (decarboxylation),
alkali (oxidative ring fission), oxygen (oxidation of
catechol units), and hydrogen peroxide, even during
their (bio)synthesis. They also undergo alteration with
physical agents such as heat and light, as well as with
aging, even if left dry on a shelf.
2. Melanins vary their properties with the degree of
hydration. Dry melanins (especially eumelanins) are
tightly aggregated and may exhibit physical properties
that are different from those of freshly collected wet
samples.
Consideration of these caveats was central to all
experimental protocols reported in this paper for struc-
tural and biological studies of melanins.
The impact of the isolation procedure on the structural
and physical properties of natural melanins has been
addressed earlier (Liu and Simon, 2003). Three important
issues emerged, namely: (i) the close association of
proteins and other biological components with melanin;
(ii) the variety of metal cations present in the natural
pigment; and (iii) the influence of the drying method on
the physical properties of the pigment, for example
aggregation state, surface area-to-mass ratio, and poros-
ity of the material.
The presence of tightly bound cellular components is
the major obstacle to the isolation of melanins from
natural sources. Harsh hydrolytic treatments with boiling
mineral acids or alkali have been abandoned following
realization that, in spite of the lack of visual changes,
pigment skeleton and functionalities are profoundly
affected. Heating of melanins with or without hydrochlo-
ric acid at reflux has been shown to lead to extensive
617
d’Ischia et al.
decarboxylation (Ito, 1986; Prota, 1992), and care must be
taken to avoid extremes in pH and temperature. In
general, no attempt should be made to separate melanin
from these internal proteins, because such a separation
would destroy the granules. Current strategies focus on
the attack of the keratin matrix by breaking disulfide
bonds and exposure to proteolytic digestion (Novellino
et al., 2000).
Physical disaggregation of particulate melanin using a
wet milling step was also found to facilitate the removal
of significant quantities of adsorbed protein and other
molecular structures in various forms. Issues are posed
by the large number of free carboxylic acid residues in
natural melanins, responsible for the cation-exchange
properties.
Methods for purifying melanin in the form of intact
melanosomes have been reported (Watabe et al., 2005).
These methods utilize highly pigmented cells to maximize
recovery and employ sucrose density gradients to sepa-
rate melanosome fractions based on their density (which is
determined in large part by the amount of melanin pigment
that they contain). See also Appendix S1 pages S3–S5.
Sepia melanin
Sepia ink has been widely investigated as one of the most
accessible natural sources of eumelanin, and a variety of
isolation procedures have been described (Liu and Simon,
2003, 2005; Simon et al., 2008). Iterative washings of the
ink with water and centrifugation give a pellet containing
largely spherical granules (150 nm in diameter) with a
protein content of 6–8% by mass. A very simple isolation
procedure that would not affect pigment integrity involves
centrifugation of freshly collected ink in 0.01 M HCl in the
cold, followed by storage at 4°C without desiccation and in
the absence of oxygen (Pezzella et al., 1997). The pigment
thus obtained usually shows a protein content of approx-
imately 5%. Sepia melanin has been a proposed standard
for natural eumelanin (Chedekel et al., 1992).
Hair melanin
Pigments from hair have been widely used as a model for
human melanin. Melanin in human hair is deposited in the
form of granules in the medulla, in the porous core of
the hair fiber, and in the cortex surrounding it. Access to
the inside of the hair by a proper swelling of the
overlapping layers of the external covering, the cuticle,
is therefore necessary for pigment release. By sequential
use of proteinase K, papain and protease, and pretreat-
ment of the tissue with dithiothreitol, protein removal can
be efficient. The enzymatic extraction preserves the
integrity of the melanosome, removes most of the
external proteins, and therefore should be the preferred
choice for the isolation of melanin from hair samples (Liu
et al., 2003; Novellino et al., 2000).
Homogenization of the finely minced hair sample is
required prior to exposure to proteolytic agents to favor
their action. This can be achieved by the use of a glass/glass
618
potter such as Tenbroeck homogenizer, while other
mechanical devices (grinder, ultrasonic disrupters) cur-
rently employed for tissue homogenization prove often
inappropriate. It is preferable to use freshly collected hair
samples, as photoaging induces structural modifications
not only in pheomelanins (Greco et al., 2009) but also in
eumelanins (Wakamatsu et al., 2012a).
Retinal pigment epithelium (RPE) melanosomes
In some cases, pigmented cells can be lysed and intact
melanosomes can be isolated from other cellular constit-
uents, for example RPE. Ultracentrifugation on a discon-
tinuous sucrose gradient yields high-purity samples
where the integrity of the melanosomes is preserved.
This is exemplified by the atomic force microscopy (AFM)
images of bovine RPE melanosomes isolated in this
manner (Liu et al., 2005).
Iridial melanosomes
Treatment of the iridial tissue with collagenase results in a
substantial removal of the protein as the result of a specific
attack to collagen, which cannot be achieved with other
proteolytic enzymes, such as those used for hair. Non-
collagenic protein structures can be specifically attacked
by trypsin, while lipids and glycoproteins may be removed
by pancreatin. The protocol ensures an efficient protein
removal without detectable modification of pigment
structure (Novellino et al., 2000; Peles et al., 2009).
B16 mouse melanoma
Intact melanosomes from B16 melanoma can be
obtained by sucrose density gradient ultracentrifugation
followed by the treatment of the pigment fraction with
Triton and SDS in 0.1 M Tris–HCl, pH 7, to remove
strongly retained impurities. Extensive washings give a
pigment that can be used for structure determination
purposes (Jimbow et al., 1984). The purity of melano-
somes can be checked under electron microscopy after
fixing melanosome pellets with 1% osmium tetroxide. An
electron micrograph showing the ultrastructure of the
starting material and a purified melanosomal preparation
is provided in Appendix S1.
Feather melanin
Most isolation protocols involve the use of alkaline
conditions (0.1 M NaOH) to disrupt the hard keratin
matrix (Prota, 1992) and cause the release of the yellow
brown pigment in solution. Actually, the mild repeated
enzymatic treatment used for hair samples may be likely
extended to these tissues provided that an efficient
homogenization of the sample is previously performed.
Neuromelanin
Human neuromelanin differs from other melanins in that
it consists of a pheomelanin core generated from cystei-
nyldopamine and wrapped by a eumelanin-like dopamine-
derived shell (Bush et al., 2006). The isolation and
ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
 Methods in melanin research

purification of neuromelanin is very complex due to the
presence of the tissue matrix that tightly retains the
pigment. Human neuromelanin contains as main impuri-
ties glycolipids, dolichol and chemically bound metals
such as iron and zinc, and proteins (Fedorow et al., 2005;
Wakamatsu et al., 1991; Zecca et al., 2001). Accordingly,
recommended protocols for the preparation of substantia
nigra neuromelanin involve a complex sequence of steps,
including homogenization of tissues, washings with
phosphate buffer to remove water-soluble components,
treatment with SDS and proteinase K to remove proteins,
dialysis to remove inorganics and other low molecular
weight species, and finally washing with methanol and
hexane to remove lipid components (Zecca et al., 2004).
dopachrome, a red-colored, reactive intermediate in
eumelanogenesis. When a neutral solution of dopa-
chrome, prepared by ferricyanide oxidation of DL-dopa, is
kept at neutral pH, decarboxylation prevails and DHI is
by far the main product (Wakamatsu and Ito, 1988; see
Appendix S1 page S6). When the pH of the dopachrome
solution is raised to 13, DHICA is rapidly produced
instead through tautomerization. Both dihydroxyindoles
are separated by extraction and purified by crystalliza-
tion, to give almost colorless crystals in good yields
(60–80%). Although these dihydroxyindoles, especially
DHI, are extremely labile to auto-oxidation, they are
rather stable when kept in a deep freezer (sealed under
argon).

Melanin precursors Cysteinyldopas and cysteinyldopamines

Commercially available melanin precursors include 5-S-Cysteinyldopa (5SCD) is regarded as the main bio-
L-tyrosine and L-dopa, as well as dopamine and related synthetic precursor of pheomelanins. 5SCD is obtained as
catecholamines (for comments on dopamine-derived main product by the reaction of dopaquinone, generated
melanin, see following section). Non-commercial precur- by chemical or enzymatic oxidation of dopa, with excess
sors include 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole- cysteine. Small amounts of other cysteinyldopa isomers,
2-carboxylic acid (DHICA), and cysteinyldopas (Figure 1). namely 2-S-cysteinyldopa (2SCD), 6-S-cysteinyldopa, and
These may be used to prepare model melanin pigments 2,5-S,S-dicysteinyldopa, are also formed (Chioccara and
for structural and functional studies. Although multistep, Novellino, 1986; Ito and Prota, 1977).
gram-scale syntheses of 5,6-dihydroxyindoles and cys- At present, there are two one-pot procedures to obtain
teinyldopas have been reported, we restrict the present 5SCD from subgram to gram scale, involving the gener-
discussion to expedient subgram-scale preparations use- ation of dopaquinone by the oxidation of dopa followed by
ful for non-chemists and that do not require organic rapid reaction with cysteine. Procedure (i) involves the
synthesis skills nor special laboratory equipments. Criteria use of cerium ammonium nitrate in 2 M sulfuric acid to
for purity are provided for each compound (HPLC, oxidize L-dopa and the resulting dopaquinone is poured
elemental analysis, UV, NMR, MS). into a solution of L-cysteine in 2 M sulfuric acid under a
flux of argon. Biomimetic-type procedure (ii) involves the
5,6-Dihydroxyindoles use of mushroom tyrosinase (and O2) as an oxidant and
The preparation of DHI and DHICA in subgram quanti- the resulting dopaquinone is trapped by the cysteine
ties takes advantage of the chemical behavior of present in the solution (Ito and Prota, 1977) (see also
Appendix S1 pages S7–S13). Fractionation of the reaction
HO NH2 mixture affords 5SCD in moderate to good yields (40–
HO NH2
65%), while 2SCD is obtained in lower yields (approxi-
COOH mately 10%). Another gram-scale preparation of 5SCD
HO HO dopamine
(and 2SCD) was reported using hydrogen peroxide in the
dopa presence of iron–EDTA complex as an oxidizing agent
HO (Ito, 1983).
HO
COOH 2SCD, the minor constituent of red hair pheomelanin
HO N (Greco et al., 2009; Wakamatsu et al., 2009), can be
HO N H
H 5,6-dihydroxyindole- selectively prepared by a gram-scale multistep synthesis
5,6-dihydroxyindole 2-carboxylicacid (DHICA) starting from commercially available caffeic acid (Panzella
(DHI)
et al., 2007b).
HOOC NH2 5-S-Cysteinyldopamine and its isomer 2-S-cysteinyl-
HO NH2
dopamine are regarded as minor precursors of neuromel-
COOH S anin (Wakamatsu et al., 2003) and can be prepared by
HO tyrosinase oxidation of dopamine in the presence of
HO NH2
S cysteine (Ito et al., 1986).
COOH
H2N COOH HO
Synthetic melanins
5-S-cysteinyldopa (5SCD) 2-S-cysteinyldopa (2SCD)
Main types of synthetic melanins include eumelanins,
Figure 1. Chief melanin precursors. pheomelanins, and neuromelanins, the latter modeled by

ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 619
d’Ischia et al.
the oxidation of dopamine in the presence of cysteine
(Wakamatsu et al., 2003, 2012b). Their preparation
should be based on conditions expected to mimic at best
melanogenesis in the biological environment. However, a
broad variety of methods and procedures have been
described, involving diverse parameters that do not
always meet the requisite of biological relevance. Some
representative procedures reported in the literature for
the preparation of synthetic eumelanins are schematically
summarized in the Appendix S1 section. Oxidation of
dopamine, originally investigated as a model approach to
neuromelanin, has recently become popular as a method
for preparing a dark eumelanin-like material known as
polydopamine, with extraordinary adhesion and coating
properties (Lee et al., 2007). Polydopamine possesses
structural and physicochemical properties in common
with eumelanins, and thus, it may be also referred to as
dopamine melanin. It has been shown to consist of both
cyclized (DHI) and uncyclized units in variable proportions
depending on the mode of preparation (Della Vecchia
et al., 2013) and is therefore different from pure DHI-
melanin in that it contains non-cyclized catecholamine
units.
When preparing synthetic melanins, three important
sets of parameters must be considered:
1. Precursors.
2. Reaction conditions (e.g., oxidants, medium, pH,
reaction time).
3. Post-synthetic procedures, including work-up (precip-
itation, reduction, centrifugation, lyophilization) and
storage (dry, cold, under nitrogen).
Structural features and properties of synthetic melanins
depend on all of the above factors. Therefore, it may be
misleading to compare pigments prepared under different
conditions even if from the same substrate.
Eumelanins
Precursors
Main precursors used to prepare synthetic eumelanins
include L-tyrosine, L-dopa, DHI, and DHICA. Tyrosine and
dopa-melanins consist largely of DHI-related units with
some 10% of DHICA, whereas natural eumelanins
contain DHI and DHICA units in approximately 1:1 ratios
(Ito, 1986; Pezzella et al., 1997). Commonly used proto-
cols for the preparation of polydopamine or dopamine
melanin are based on prolonged aerial oxidation of the
catecholamine (10 mM) in Tris or other buffers at pH 8.5
leading to black insoluble materials (Della Vecchia et al.,
2013).
Reaction conditions
A suitable precursor concentration to mimic the in vivo
situation is 1 mM (the natural substrate L-tyrosine is not
soluble above 2.5 mM). However, for the practical
reason of preparing subgram quantities of melanins,
10 mM concentration is preferable. Varying this parameter
620
may affect the degree of polymerization and aggrega-
tion. Tyrosinase is the oxidizing system of choice,
although caution may be in order against the use of
the commercial mushroom enzyme, largely because of
its low specificity and different mode of action from the
mammalian enzyme. Because tyrosinase is inactivated
during the oxidation (Land et al., 2007), its concentration
should be such to ensure complete substrate consump-
tion without total inactivation. Peroxidase/hydrogen
peroxide provides an alternate system for the prepara-
tion of synthetic melanins from DHI and DHICA (d’Ischia
et al., 2005). Under these conditions, pigment formation
proceeds rapidly and the yields are higher than with
tyrosinase. Care should be taken to use the minimal
amount of hydrogen peroxide to allow for a complete
oxidation of the substrate without concomitant degra-
dation of the pigment as formed. This amount can be
fixed in two molar equivalents with respect to the
substrate.
Air flux (e.g., simple stirring or shaking) in the place of
oxygen is recommended to ensure a more biomimetic
and reproducible oxygen tension during polymerization
and a lower competition of auto-oxidative and peroxida-
tive processes. Chemical oxidants that can be used
include potassium ferricyanide or periodate, but their
amounts and general use are subject to caveats depend-
ing on the scope of the synthesis and whenever a truly
biomimetic procedure is required.
A suitable medium is phosphate buffer at 0.05–0.1 M
concentration and at pH around neutrality (6.8, 7.0, or
7.4). Use of alkali (pH > 8) should be avoided whenever a
biomimetic material is desired, due to profound structural
alterations secondary to quinone ring fission (Prota,
1992).
Post-synthetic procedures
Work-up is typically based on precipitation with dilute
acids, which serves the dual scope of stabilizing catechol
components of the pigment to further oxidation and
favoring collection. Drying may be carried out either in a
desiccator or by lyophilization, with the latter being
preferable whenever profound physical alterations
caused by extensive desiccation are not desired (see
General properties).
Pheomelanins
Precursors
Synthetic pheomelanins may be prepared by the oxida-
tion of dopa in the presence of cysteine, or by the
oxidation of 5SCD. The former protocol gives a more
biomimetic pigment because it may contain all of the
cysteinyldopa isomers produced by the dopa-cysteine
reaction, while the latter gives a chemically more
homogeneous standard that may be useful to investi-
gate specific structural aspects (Wakamatsu et al.,
2009).
ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
 Methods in melanin research

3-AHP
HI Hydrolysis

24.7
Reaction conditions

ND
ND
ND
ND

ND
ND
0.0
A suitable concentration for L-dopa and L-cysteine or for

(lg/mg)

4-AHP
5SCD would be 1 mM. However, for preparative pur-

148
373
ND
ND
ND
ND

ND
ND
poses, 10 mM of both L-dopa and L-cysteine ensures high
product yields and recovery (Wakamatsu et al., 2009).

TDCA
Use of excess cysteine is no longer recommended

0.0
0.0
0.0
0.0
6.6
7.7

0.0
0.0
because the intermediate cysteinyldopas are oxidized
too slowly to pheomelanin unless a trace of dopa acts as

H2O2 oxidation (lg/mg)

TTCA

0.0
0.0
0.0
0.0

0.0
0.0
13.6
16.6
a catalyst (Ito, 1989).
It is still unresolved whether there is as yet undis-
covered enzymology in the synthesis of pheomelanins.

PDCA
Tyrosinase may be used as an oxidizing system for

4.4
2.5
1.4
0.5
1.2
1.3

1.8
0.6
pheomelanin synthesis (for comments on enzyme
concentration, see text above concerning eumelanins),

PTCA

40.1

16.4
6.2
2.5

5.3
8.1

1.5
106.2
although oxidation of 5SCD requires L-dopa (0.05 eq.) as
a catalyst. Again, air flux is recommended instead of

(A500/mg)
oxygen to provide a more biomimetic oxygen tension
and to minimize auto-oxidative and peroxidative

6.97

8.49
4.06
4.26
5.73

4.81
5.96
10.60
TMa
processes. Oxidation of cysteinyldopas can also be
carried out with peroxidase/hydrogen peroxide (Panzella

Elemental composition (per N)

et al., 2010). Addition of Zn2+ ions markedly affects the
synthetic process and is recommended whenever more
homogeneous preparations with a higher benzothiazine

C11.23H12.93N2S0.95O6.37
C11.51H13.08N2S0.95O6.17
content are desired. The role of Zn2+ ions is to direct
the reaction course toward the formation of carboxyl-

C7.57H6.78N1O4.72
C7.78H6.04N1O3.96
C8.09H6.43N1O5.15
C8.41H7.94N1O6.76

C8.36H5.97N1O5.72
C7.61H5.75N1O6.59
ated benzothiazine units. Phosphate buffer at 0.05–
0.1 M concentration and at pH 6.8–7.4 is a suitable
medium.

Post-synthetic procedures
mg
mg
mg
mg
mg
mg
Typical work-up is based on precipitation at pH 3 with
Yield

acetic acid. Lower pH values would render pheomelanin

156
175
191
211
201
127
soluble.
Tyrosinase

U
U
U

U
U

U
Commercial eumelanins
50,000
50,000
50,000

50,000
100,000

100,000
Table 1. Preparation and analysis of synthetic and commercial melanins

A few companies produce synthetic eumelanins for

research purposes. In 2012, Sigma catalogue offers a
product labeled melanin (CAS-No. 8049-97-6, EC-No.
L-Dopa 1 mmol + L-cysteine 1 mmol

232-473-6), which is reported to be prepared by the

DHI 0.5 mmol + DHICA 0.5 mmol

oxidation of tyrosine with hydrogen peroxide. The

Peroxide oxidation of tyrosine
5SCD 1 mmol + L-0.05 mmol

product differs from natural eumelanins in that it is

freely soluble in slightly alkaline aqueous medium.
From Sepia officinalis

Considering the marked difference between the tyro-

sine–hydrogen peroxide reaction and the biogenetic
DHICA 1 mmol

origin of natural melanin, use of the commercial syn-

Dopa 1 mmol
DHI 1 mmol
Description

thetic melanin should therefore be subject to caution.

Comparison of various properties of synthetic eumela-
nins, commercial melanins, and sepia melanin is
reported in Table 1.
DHI+DHICA-melanin

Under the same CAS number, a melanin from Sepia

Dopa+Cys-melanin

officinalis is also offered. Different preparations of sepia

Tyrosine-melanin
DHICA-melanin

5SCD-melanin

Sepia melanin
Dopa-melanin

melanin were found to give similar analytical values

Total melanin.
DHI-melanin

(Table 1). This sepia melanin may be used as a

Commercial
Synthetic

reference standard (with lot number) for characterization

Melanin

by chemical degradation (see below) and UV/VIS

absorbance.
a

ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 621
d’Ischia et al.

3-amino-4-hydroxyphenylalanine (3-AHP) (Wakamatsu

Analytical methods
et al., 2002). 4-AHP can be prepared by HI hydrolysis of
Marker preparation 5SCD-melanin (Wakamatsu et al., 2009). However, this
Several markers are currently used for melanin analysis amino acid can be more readily obtained in a 100 mg
but only two, pyrrole-2,3,5-tricarboxylic acid (PTCA) and 3- quantity by the nitration of commercially available
amino-4-hydroxyphenylalanine (3-AHP), are commercially m-tyrosine followed by HI reduction of the resulting
available. Thus, most markers must be prepared by 3-hydroxy-4-nitrophenylalanine (along with other possible
simple chemical processes, as reported below. The purity isomers). Likewise, 4-amino-3-hydroxyphenylethylamine
of the markers described in this section was assessed by (4-AHPEA) and 3-amino-4-hydroxyphenylethylamine
HPLC, elemental analysis, UV, NMR, and MS analyses (3-AHPEA), degradation products of neuromelanin, can
(see Appendix S1). be obtained by HI hydrolysis of 5-S-cysteinyldopamine-
Typical degradation markers for eumelanin analysis and melanin and 2-S-cysteinyldopamine-melanin, respectively
quantitation are pyrrole-2,3,5-tricarboxylic acid (PTCA) and (Wakamatsu et al., 2003).
pyrrole-2,3-dicarboxylic acid (PDCA) (Figure 2), which can Alkaline hydrogen peroxide degradation of pheomelanin
be readily obtained in 100 mg quantities by oxidative yields 6-alanyl-2-carboxy-4-hydroxybenzothiazole (BTCA),
degradation of commercial 5-hydroxyindole-2-carboxylic 7-alanyl-2-carboxy-4-hydroxybenzothiazole (BTCA-2), thia-
acid and 5-hydroxyindole, respectively, followed by zole-2,4,5-tricarboxylic acid (TTCA), and thiazole-4,5-dicar-
extraction and crystallization. (Ito and Wakamatsu, boxylic acid (TDCA). BTCA and BTCA-2 can be obtained
1998). Recently, pyrrole-2,3,4,5-tetracarboxylic acid by alkaline hydrogen peroxide oxidation of 5SCD-melanin
(PTeCA) and pyrrole-3,4,5-tricarboxylic acid (isoPTCA) and 2SCD-melanin, respectively (Greco et al., 2009;
were added to a list of oxidation products from eumelanin Napolitano et al., 1996). BTCA can be easily prepared
(Ito et al., 2013a,b). Those pyrrole carboxylic acids appear from dopa and cysteine through a one-pot, 4-step
to derive from cross-linking of dihydroxyindole moiety procedure involving the use of ferricyanide and Zn2+ to
that occurs during (photo) aging. induce oxidative conversion of intermediate 5SCD into 3-
Several pheomelanin degradation products can be used carboxybenzothiazine, and of acidic persulfate to promote
as markers for pigment determination in tissues ring contraction. Purification of the final mixture by HPLC
(Figure 2). 4-Amino-3-hydroxyphenylalanine (4-AHP) is a gives BTCA in around 50% isolated yield. Starting from
major degradation product of pheomelanin upon hydroi- pure 5SCD, the procedure is reduced to three steps plus
odic acid (HI) hydrolysis, along with the minor isomer chromatographic purification. This latter procedure,
applied to 2SCD, allows for the preparation of BTCA-2.
6-Alanyl-4-hydroxybenzothiazole (BT) and 7-alanyl-4-
COOH HOOC hydroxybenzothiazole (BT-2) may be obtained by the
same protocol with a final thermal decarboxylation step
COOH HOOC COOH preceding HPLC fractionation (Greco et al., 2009). TTCA
N N
H H
and TDCA are obtained similarly from 5SCD-melanin
PDCA PTCA
(Wakamatsu et al., 2003). TTCA can also be obtained in
good yield as the triethyl ester by a simple chemical
HO NH2 H 2N NH 2
synthesis from commercially available ethyl thiooxamate
R R and diethyl chlorooxaloacetate (von Erlenmeyer et al.,
H 2N HO
1948) with some modification. Alkaline hydrolysis of the
4-AHP (R = COOH) 3-AHP (R = COOH)
4-AHPEA (R = H) 3-AHPEA (R = H) triethyl ester affords tripotassium salt of TTCA, used as
the standard. Decarboxylation of TTCA under controlled
OH
OH acidic conditions yields TDCA in good yield.
N
N
R
R Chemical degradation
S
S
Chemical oxidation of eumelanins under various condi-
H2N
H 2N COOH tions gives PTCA as a major product (Prota, 1992), which
COOH serves as a basis for quantitative analysis of eumelanins.
BTCA, R = COOH
BT, R = H BTCA-2, R = COOH
PTCA is regarded also as a specific index of DHICA units
BT-2, R = H or 2-substituted DHI units, whereas PDCA levels indicate
HOOC N 2-unsubstituted DHI units. The first microanalytical appli-
R cation of the reaction was based on HPLC analysis of
HOOC S PTCA produced upon permanganate oxidation of eumel-
anins in 1 M sulfuric acid (Ito and Fujita, 1985), which
TTCA, R = COOH became a standard of eumelanin assay for some time (Ito
TDCA, R = H
and Wakamatsu, 2003). However, a number of disadvan-
Figure 2. Main melanin markers. tages prompted replacement with an alternative method

622 ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

involving hydrogen peroxide oxidation in 1 M NaOH
(Napolitano et al., 1996, 2000) or in 1 M K2CO3 (Ito and
Wakamatsu, 1998; Ito et al., 2011b; Pezzella et al., 1997).
Advantages of the latter method include (i) omission of an
extraction step with ether, (ii) production of PDCA from
DHI-derived units, (iii) higher yields of PTCA, and (iv)
concomitant production of pheomelanin markers BTCA,
BTCA-2, TTCA, and TDCA that can be analyzed simulta-
neously (Ito et al., 2011b). While the alkaline hydrogen
peroxide degradation is definitely the method of choice
for eumelanin characterization, which of the two variants
based on 1 M NaOH or 1 M K2CO3 as a medium is better
remains a matter of convenience. With 1 M NaOH
artificially produced interfering peaks are more obvious
because of the harsh conditions, while yields of BTCA and
BTCA-2 are greater than with 1 M K2CO3 (Ito et al.,
2011b). Analytical conditions, particularly the HPLC
mobile phase, are also critical for satisfactory elution
and reliable identification and quantitation of the melanin
markers.
Pheomelanin degradation products used in current
analytical methods include 4-AHP, 3-AHP, TTCA, TDCA,
BTCA, and BTCA-2 (Greco et al., 2009; Ito and Wakama-
tsu, 2003; Panzella et al., 2007a; Prota, 1992), while
recently identified pyridine-containing fragments await
further evaluation (Greco et al., 2011, 2012). Choice of
the best methodology depends on several factors,
including the type of sample, equipment availability, and
the researcher’s familiarity with laboratory techniques.
The first microanalytical method was based on HI
hydrolysis of pheomelanin-containing tissues followed by
HPLC determination of 4-AHP as a marker of 5SCD-
derived units (Wakamatsu et al., 2002).
An alternate, more recent method is based on alkaline
hydrogen peroxide degradation of the sample followed by
HPLC quantitation of BTCA and TTCA (together with
PTCA) (Ito et al., 2011b; Panzella et al., 2007a). While
TTCA is a specific marker of benzothiazole units, 4-AHP,
and possibly in part BTCA, derives from benzothiazine-
containing structures (Napolitano et al., 2000; Wakama-
tsu et al., 2009). With both methods, 3-AHP and BTCA-2
can be analyzed as indices of 2SCD-derived units. The
ratios of 4-AHP to 3-AHP (HI hydrolysis) and BTCA to
BTCA-2 (alkaline hydrogen peroxide degradation) would
thus provide quantitative data about the relative propor-
tions of 5SCD/2SCD units in pheomelanins, although the
differential stability of 5SCD versus 2SCD benzothiazine
units during photochemical and thermal decomposition
must be kept into account (Greco et al., 2009; Ito et al.,
2011b; Wakamatsu et al., 2012a).
Although both 4-AHP and BTCA are produced in good
yields, 4-AHP is usually revealed with an electrochemical
detector, whereas BTCA and TTCA analyses require UV
detection. Therefore, the 4-AHP-based procedure results
in a much greater sensitivity and would be the method of
choice for detecting pheomelanins in human epidermis or
cultured melanocytes, which are available only in minute
ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Methods in melanin research
quantities. A disadvantage is that removal of HI may not
be easy in a biology laboratory. On the other hand, both
BTCA and TTCA can be selected as reliable pheomelanin
markers that provide complementary information and are
suitable for routine pigment analysis, although their
determination is affected by the lower sensitivity, due
to instrumental limitations, and, in the case of TTCA, by
some artificial production from eumelanic human hairs
(Ito et al., 2011b). A careful selection of chromatographic
conditions may also be critical. Selection of BTCA or
TTCA as marker may thus be dictated by considerations
relating to synthetic access, structural specificity, and
analytical properties.
A comparative view of the various analytical methods
applied to the characterization of synthetic melanins
described above is given in Table 1.
Spectral and biophysical studies
EPR spectroscopy
Although EPR spectra of melanins are quite characteris-
tic, they are by no means unique to melanin. This is
particularly true for eumelanins. However, the unusual
physicochemical properties of melanin pigments make
their identification based on responses of their EPR signal
to selected agents relatively unambiguous (Sealy et al.,
1982). Especially relevant is the so-called comproportion-
ation equilibrium, that is, the equilibrium between fully
reduced (H2Q), fully oxidized (Q) melanin units, and their
semi-reduced (semi-oxidized) forms (SQ) (Felix et al.,
1978; Sarna and Plonka, 2005). It can be described by a
simple equation:
H2 Q þ Q ! SQ þ 2Hþ
In eumelanins, the reduced units are most likely of DHI
or DHICA nature, while the oxidized units are the
corresponding indolequinones. Of course, the semi-
reduced (semi-oxidized) units are the so-called extrinsic
melanin free radicals. The comproportionation equilibrium
in melanins can be modified by many factors including
pH, light, redox agents, and diamagnetic multivalent
metal ions. The intensity of the melanin EPR signal and
its anisotropy increases with pH and doping the melanin
with zinc ions. At X-band (microwave frequency approx-
imately 9 GHz), at which standard EPR spectrometers
operate, EPR spectra of eumelanin represent a single
slightly asymmetric line 0.4–0.6 mT wide with a g-factor
close to 2.004. For fully hydrated samples, the anisotropy
of the melanin EPR signal and its g-factor are a function of
pH. Pheomelanin has a broader signal with the total width
approximately 3 mT and g = 2.005. The EPR signal of
pheomelanin is reminiscent of immobilized nitroxide EPR
spectra with distinct hyperfine splitting, except the latter
exhibit larger hyperfine splitting than the former. At
comparable concentration, the EPR signal amplitude of
623
d’Ischia et al.
pheomelanin is about one order of magnitude lower than
that of eumelanin (Figure 3).
The observable EPR signal intensity of melanin can be
modulated by an order of magnitude and can also be
enhanced by steady-state irradiation with visible and near
UV light. The effect is transient, and after termination of
the irradiation, the signal intensity slowly returns to its
initial level if no photo-oxidation occurred. Removal of
oxygen from the samples prior to their irradiation may
therefore be appropriate. It is important to stress that
comproportionation equilibrium of the melanin subunits,
responsible for the observable changes in the melanin
EPR signal intensity, only operates in fully hydrated
melanin. It has been shown that deep dehydration of
melanin irreversibly modifies physicochemical properties
of the pigment, including susceptibility of its EPR signal to
changes in pH, doping with zinc ions, and irradiation with
light. It appears that excessive drying of melanin abates
its comproportionation equilibrium (Mostert et al., 2012a;
Sealy et al., 1980).
The effect of a significant change in the compropor-
tionation equilibrium of the melanin subunits induced by
saturating melanin with zinc ions is shown in Figure 4. It
is apparent that the same concentration of eumelanin and
pheomelanin in the presence of high concentration of zinc
ions exhibits EPR signals with intensities over one order
of magnitude larger than those observed in PBS (see
Figure 3 for comparison). In addition, complexation of
zinc ions makes the pheomelanin component significantly
more pronounced both in samples containing synthetic
cysteinyldopa-melanin (Figure 4B) and in melanoma cells
(Figure 4C). Arrows in Figure 4 indicate the eumelanin
and pheomelanin components of the pigment examined.
Because eumelanin is a good chelator of multivalent
transition metal ions (Meredith and Sarna, 2006) and
metal ions bound to eumelanin can modulate the intensity
of the observable EPR signal of melanin radicals (Sarna
et al., 1976), quantitative determination of eumelanin by
EPR spectroscopy may require preliminary removal of
bound metal ions. This can be achieved by prolonged
washing of the samples with aqueous solutions of strong
chelators such as EDTA, DTPA, and desferrioxamine at
high concentration (Enochs et al., 1993; Shima et al.,
1997; Zecca et al., 2008). An alternative approach is to
acidify melanized samples with strong acids – H2SO4 or
HCl – to pH 0–1. It is believed that such a treatment will
release most of the bound metals. Of course, the melanin
A B C
624
standard should be treated the same way, and the risk of
pigment degradation cannot be neglected.
If room temperature EPR examination is preferred or
required, aqueous suspension of the melanized material
is transferred to standard quartz EPR flat cells (approxi-
mately 0.3 mm internal thickness and 8 mm width).
Considering that lower amount of fully hydrated material
can be examined at room temperature compared to liquid
nitrogen temperature, the corresponding lower Q factor
of the resonant cavity at room temperature and different
microwave power saturability of the melanin EPR signal
under both experimental conditions, it is safe to conclude
that the room temperature EPR examination of melanin is
several times less sensitive than that performed at liquid
nitrogen temperature. In addition, the proper position of a
flat cell with aqueous sample in the resonant cavity is
critical for its correct tuning, making quantitative deter-
mination of lossy samples at room temperature more
difficult than at liquid nitrogen temperature.
Optical, electrical, and microstructural
characterization
A detailed understanding of the optical, electrical, and
microstructural properties of melanin not only provides
another tool to define and identify melanins, but also
opens the intriguing possibility of melanin bioelectronics
(Bothma et al., 2008; d’Ischia et al., 2009).
Typically, the optical absorption of a well-dispersed
melanin suspension is monotonic, broad, and featureless
when Mie scattering is eliminated or accounted for (Riesz
et al., 2006). The spectrum has an exponential depen-
dence upon wavelength (or sigmoidal with respect to
energy). A typical solution absorption spectrum of syn-
thetic melanin formed from the auto-oxidation of dl-dopa
in alkaline aqueous solution is provided as Appendix S1
(Meredith et al., 2006). Solid eumelanin pellets, aggre-
gated solutions, or thin films often show pronounced Mie
scattering which broadens, flattens, and extends the
absorption into the near infrared. An integrating sphere
must be used to collect such spectra in reflectance or
transmittance to negate these effects, and recover the
‘real’ spectrum. Solid eumelanin thin films suitably disag-
gregated have almost identical absorption spectra to well-
dispersed solutions (Bothma et al., 2008). This indicates
that the fundamental absorption of melanins results from
the primary chromophore and not some secondary
aggregated state. Such results have also been reproduced
Figure 3. EPR spectra of 0.9 mg/ml
enzymatic dopa-melanin (A), 0.2 mg/ml
enzymatic cysteinyldopa-melanin (B), and
human melanoma cells (C). All samples
were suspended in phosphate-buffered
saline (PBS) and examined at 77K using the
same apparatus parameters.
ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

A B
Figure 4. EPR spectra of dopa-melanin (A), cysteinyldopa-melanin (B), and human melanoma cells (C) in the presence of 50 mM zinc acetate.
Samples contained the same amount of melanin or cells as in Figure 3 and were run under identical experimental conditions. Solid arrows indicate
the low-field component of eumelanin EPR signal, and broken arrows show low-field and high-field components of pheomelanin EPR signal.
with eumelanin thin films synthesized in, and cast from,
alternative solvents such as dimethylsulfoxide (DMSO)
(Abbas et al., 2011). Polymerization of melanin from
DHICA or tyrosine produces ‘peaks’ in the monotonic
absorption profile, possibly a signature of residual, low
molecular weight precursor in the system, or residual
protein in purified natural melanins (Tran et al., 2006).
Although generally considered to be non-fluorescent,
eumelanin does emit radiation when photostimulated,
albeit with a tiny quantum yield of order 104. Further-
more, the emission conforms to that expected from a
typical organic chromophore (Appendix S1) and is excita-
tion energy dependent with a complex signature charac-
teristic of an ensemble of multiple chromophores
(Meredith et al., 2006) according to the idea of ‘chemical
disorder’. Such measurements require extremely sensi-
tive emission detection and careful experimental tech-
nique (particularly in relation to accounting for so-called
inner filter effects and emission re-absorption) (Riesz
et al., 2005).
In the solid state (i.e., thin films or pressed powder
pellets), eumelanin conducts electricity. Its electrical
properties have a strong dependence upon its state of
hydration, the form of which is critically dependent upon
how the measurement is made and whether the system
is allowed to reach equilibrium with its environment.
Measurements of electrical conduction in a so-called
sandwich electrode configuration (see Appendix S1)
produced erroneous results due to the limited sample
surface area available to absorb water (Mostert et al.,
2012a). This non-equilibrium behavior delivers a hydration
dependence consistent with the so-called modified
dielectric theory of Powell and Rosenberg (Powell and
Rosenberg, 1970) – a fact used to justify the now
debunked model of melanin as a natural amorphous
semiconductor (Mostert et al., 2012b). However, it has
recently been demonstrated that the hydration depen-
dence of the DC electrical properties of eumelanin has a
completely different functional form if careful equilibrium
measurements are made with knowledge of the water
adsorption isotherms (Mostert et al., 2010, 2012a). This
delivered strong evidence that eumelanin is a hybrid ionic-
electronic material dominated by the flow of protons as
the primary charge carrier. Absorbed water locally titrates
ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Methods in melanin research
C
the comproportionation equilibrium reaction generating
semiquinone species and free protons to be conducted
through the hydrated matrix via the Grotthus mechanism
(Mostert et al., 2012b).
Photoconductivity measurements support this view of
eumelanin electrical conductivity. In the solid state,
photocurrent is produced when UV or white light is
incident upon eumelanin. This photocurrent is derived
from the same mechanism as the DC electrical conduc-
tivity (i.e., chemical self-doping). Photoexcitation also
drives the formation of semiquinone radicals and water
is needed to mediate the process – hence, only wet
melanin photoconducts (see Appendix S1) (Mostert et al.,
2012b).
Hence, electrical measurements on solid eumelanin
require a detailed knowledge and control of the hydration
state of the material. Furthermore, the appropriate
electrode geometry (surface contacts versus sandwich
contacts) must be used with full knowledge of the
implications of proton-dominated electrical physics
(non-ohmic behavior, AC conductivity, and long RC time
constants) (Jastrzebska et al., 1998).
Low-contrast transmission electron microscopy (TEM)
combined with radial Fourier analysis has been used to
show that synthetic and natural melanins form a stacked
secondary structure with characteristic interplane spacing
of approximately 3.7  A. Synthetic melanins curl into
stacked ‘onion-like’ structures with relatively small
primary sheet (oligomer) dimensions (see Supplementary
Information), while natural eumelanins (sepia and bovine
epithelium) tend to form much larger sheets, but still
retain the same characteristic interplane spacing (Watt
et al., 2009). The system can be disaggregated using a
mild base and/or a p-stack breaker (Bothma et al., 2008;
Watt et al., 2009). If this procedure is performed
correctly, then the system does not bleach and the usual
melanin monotonic absorption is retained, showing again
that the optical properties are derived from the primary
structure as indicated previously.
Enzymes for in vitro studies
As opposed to the well-defined and relatively simple
reaction media employed in the chemical synthesis of
625
d’Ischia et al.
model melanins, the melanosomal lumen where the
biosynthesis of melanin pigments takes place is a
complex environment. Its dynamic nature is underscored
by the pheomelanin switch whereby a given melanocyte
switches the pattern of pigment production from eumel-
anogenesis to pheomelanogenesis (Barsh, 2006; Ito and
Wakamatsu, 2003, 2011). Thus, synthesizing models
resembling as closely as possible natural melanins
requires an understanding of the biochemical and phys-
icochemical variables that may have an impact on the final
structure of the melanin polymer. Mammalian melano-
genesis involves not only tyrosinase but also at least two
other melanogenic enzymes: tyrosinase-related protein 1
(Tyrp1 or gp75) and tyrosinase-related protein 2 (Tyrp2 or
dopachrome tautomerase, Dct). In mouse melanocytes,
Tyrp2 catalyzes the tautomerization of L-dopachrome to
DHICA and Tyrp1 acts as a DHICA oxidase. Accordingly,
the combined action of both proteins accounts for the
incorporation of DHICA to the growing melanin polymer
and for the typically higher contribution of carboxylated
indolic units in natural melanins, compared with synthetic
pigments (Aroca et al., 1990; Ito, 1986; Jime
nez-Cervan-
tes et al., 1994; Kobayashi et al., 1994; Korner and
Pawelek, 1980; Palumbo et al., 1991; Pawelek et al.,
1980). Of note, the Tyrps are expressed exclusively in
animals and are not found in prokaryotic organisms, fungi,
or plants. Importantly, the relative activity of the melano-
genic enzymes seems to determine the final structure of
the pigment, and preliminary in vitro studies indicated that
higher levels of Tyrp2/Dct yield smaller polymers of
lighter color (Aroca et al., 1992).
The different catalytic properties of the structurally
similar tyrosinase family enzymes can be accounted for
by the differential binding of specific metal cofactors.
Tyrosinase, and probably also Tyrp1, binds copper ions in
two conserved metal-binding motifs (Furumura et al.,
1998), whereas Dct/Tyrp2 is a zinc protein (Solano et al.,
1994, 1996). Because Zn ions cannot undergo redox
reactions, Dct/Tyrp2 does not function in oxidation reac-
tions, as opposed to Cu-containing tyrosinase, but instead
behaves as a tautomerization catalyst.
Extensive genetic and biochemical evidence has
shown the involvement of other melanosomal proteins
in mammalian melanogenesis. The melanosomal matrix
Pmel17 protein (also known as gp100) is the product of
the silver locus. The protein undergoes a complex
proteolytic processing within eumelanosomes. Pmel17
is required for the formation of the organelle’s lamellar
network (Hoashi et al., 2006; Kushimoto et al., 2001),
where it acts to facilitate the polymerization and deposit
of melanins (McGlinchey et al., 2009; Solano et al.,
2000). However, so far, no specific enzymatic activity
has been proposed for Pmel17. In addition to Pmel17,
other melanosomal proteins are required for the correct
maturation of the melanosomes that appears a requisite
for efficient melanization. The oculocutaneous albinism
type 1 (OA1) protein is a G-protein-coupled receptor
626
associated with the melanosomal membrane, where it
interacts with the premelanosomal protein MART-1
(Giordano et al., 2009). Inactivation of either OA1 or
MART-1 leads to the formation of structurally aberrant
melanosomes with decreased melanin contents. Other
melanosomal proteins such as the product of the under-
white locus also contribute to efficient melanin deposition
by facilitating correct trafficking and processing of tyros-
inase (Costin et al., 2003).
In addition to the melanosomal proteins and low
molecular weight thiol compounds, the pH of the melan-
osomal lumen contributes to the regulation of pigment
biosynthesis. Mammalian tyrosinases have an optimal pH
near neutrality, and the specific activity of the enzyme can
be relatively low at acidic pH (Hearing and Ekel, 1976;
Martinez et al., 1985). The melanosomal pH can also
modify the stability and the evolution of intermediates of
the melanogenic pathway, through changes in proton-
ation of ionizable groups (C
anovas et al., 1982).
Estimation of the melanosomal pH shows that the
organelle’s lumen is acidic in mouse and human mela-
noma cells or normal melanocytes (Bhatnagar et al.,
1993; Fuller et al., 2001; Puri et al., 2000), consistent
with their relationship with the endosomal–lysosomal
lineage. However, significant differences in the melanos-
omal pH have been reported for melanosomes from Black
donors, which are more neutral, and the more acidic
organelles derived from Caucasian skin (Fuller et al.,
2001).
Given the complexity of the melanosomal milieu and
the variety of components influencing the relative rates of
the melanogenic reactions, the design of biomimetic
protocols to synthesize model melanins is not an easy
task. Even the use of animal tyrosinases which appears at
first sight the simplest option to mimic human melano-
genesis is severely limited by the absence of commercial
sources of the enzyme. The purification of the mamma-
lian enzyme is technically complex and requires signifi-
cant amounts of starting tissue, most frequently
melanomas grown in mice following subcutaneous injec-
tion of a suspension of melanoma cells. A variety of
procedures have been described that most often rely on
crude preparations of melanosomes obtained by differ-
ential centrifugation as the starting material (Aroca et al.,
1990; Tomita et al., 1983), or even melanosomes purified
by density gradient centrifugation (Kushimoto et al.,
2001). Because tyrosinase is an integral membrane
protein, the melanosomal preparation must be deter-
gent-solubilized (Jime
nez-Cervantes et al., 1995) or a
soluble, enzymatically active fragment can be obtained by
limited proteolysis (Valverde et al., 1992). The resulting
extracts can be fractionated by combinations of tech-
niques such as ammonium sulfate precipitation, gel
filtration or ion-exchange chromatography, and prepara-
tive electrophoresis to obtain highly purified tyrosinase
preparations (Garcia-Borron et al., 1985; Hearing et al.,
1978; Ohkura et al., 1984; Tomita et al., 1983). However,
ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

the different purification protocols described to date are
time-consuming, and their yields are relatively low.
Another matter of concern is the possibility of subtle
differences in the kinetic behavior of the tyrosinases from
different mammalian species. For instance, it has been
shown that whereas mouse tyrosinase is devoid of
DHICA oxidase activity (Jime
nez-Cervantes et al., 1994;
Kobayashi et al., 1994), human tyrosinase is able to
oxidize DHICA to 5,6-indolequinone-2-carboxylic acid,
thus accelerating its incorporation into melanins (Olivares
et al., 2001). Overall, these limitations exclude the
general use of purified mammalian tyrosinases for the
preparation of milligram amounts of enzymatically
synthesized pigments.
A possible alternative would be the use of melanoso-
mal extracts containing the complete set of melanogenic
proteins. These extracts could be standardized by suitable
tyrosinase activity measurements and dialyzed to remove
low molecular weight thiolic compounds or metal ions
that may interfere with the metabolism of melanogenic
intermediates. Also, melanosomal extracts can be rather
easily and selectively depleted of Tyrp2/Dct activity by a
procedure that takes advantage of the much lower
thermal stability of Tyrp2/Dct as compared with tyrosi-
nase (Valverde et al., 1993). This technique might be
useful to study the impact of dopachrome tautomerase
activity in melanin structure.
Because the problems with mammalian pigmentary
enzymes are yet to be resolved, simpler methods of
enzymatic preparation of model melanins rely on com-
mercial non-mammalian tyrosinases and either mono- or
diphenolic melanogenic substrates. A highly active fungal
tyrosinase isolated from Agaricus bisporus has been used
extensively. The main advantages of this preparation are
its unlimited availability at a reasonable cost and its high
specific activity toward a variety of mono- or diphenolic
substrates. Suitable resuspensions of the commercial
powder can be used for the large-scale oxidation of
melanogenic precursors under mild conditions, and an
oxidizing environment potentially acting on melanogenic
intermediates can thus be avoided. However, these
commercial preparations should be used with care due
to several complicating factors. The fungal enzymes have
relevant kinetic differences compared with mammalian
tyrosinases. They are a relatively crude, partially purified
material containing significant amounts of other proteins,
carbohydrates, and phenolic compounds. The presence
of laccase, beta-glucosidase, beta-galactosidase, beta-
xylosidase, cellulase, chitinase, xylanase, and mannanase
can be demonstrated with suitable enzyme activity
assays (Flurkey et al., 2008; Sugumaran and Bolton,
1998). Other protein contaminants were found by protein
sequencing. Moreover, the ratios of enzymatic and
non-enzymatic contaminants may be variable. Impor-
tantly, it has been demonstrated that those contaminants
have an impact on the apparent kinetic behavior of
tyrosinase (Neeley et al., 2009) and may even lead to the
ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Methods in melanin research
misinterpretation of experimental observations (Suguma-
ran and Bolton, 1998). In addition, enzymatic preparations
from mushrooms or plant sources do not contain Tyrp1
and Tyrp2. These preparations are thus expected to
mimic poorly the distal phase of melanogenesis beyond
dopachrome and the metabolism of carboxylated indolic
intermediates.
Use of melanins and related metabolites in
biological research
There are conflicting reports on the biological role of
melanin and its intermediates, because they have been
shown to exert both toxic and antioxidant/scavenger
activities. To deal with these compounds in biological
research, it is therefore important to take account of
different parameters, that is, the specific experimental
conditions employed, the incubation time, the doses, and
the way of administration of the compounds in relation to
their solubility and stability.
Melanin precursors
Early studies demonstrated potent toxic effects of mel-
anin intermediates on different cell types. Supplementa-
tion of tyrosine in the culture medium resulted selectively
toxic to pigmented cells (Pawelek et al., 1973). Further
reports extended this evaluation on DHI, which resulted
highly cytotoxic on both melanoma cells and fibroblasts
when used at 100 lM but with no effect when admin-
istered at 10 lM (Pawelek and Lerner, 1978). DHI
appeared slightly more toxic than DHICA under experi-
mental conditions employing both long time exposure (at
concentrations of 10 and 100 lM up to 6 days) and short
time exposure (1 mM for 1 h followed by the mainte-
nance in regular medium). Estimation of the half-life of
these melanin precursors in the culture medium by HPLC
showed the shorter half-life of DHI (10–15 min) with
respect to that of DHICA (30–40 min). The unstable
nature of the intermediates in the culture medium leads
to their spontaneous auto-oxidation and the following
generation of H2O2 and other oxygen radical species, that
is, superoxide radicals. The overall cytotoxic properties of
these melanin intermediates used in cellular systems
seem therefore attributable to the production of reactive
oxygen species in the culture medium during their auto-
oxidation rather than their intrinsic cytotoxicity (Urabe
et al., 1994). The stability of DHICA up to 2 h in the
culture medium was demonstrated when the compound
was used at the concentration of 106 M or higher to
stimulate murine macrophage activity. No effects were
detected at lower or higher doses, which may be ascribed
to faster auto-oxidation of DHICA when supplemented in
culture at lower doses and to weak cytotoxic effects
when applied close to millimolar doses (D’Acquisto et al.,
1995). More recently, the protective/toxic effects of DHI
have been analyzed on eye-related cells and no cytotox-
icity was observed for the dose of 10 lM, while a
627
d’Ischia et al.
significant toxic effect resulted at 100 lM (Heiduschka
et al., 2007), in agreement with the earlier studies
(Pawelek and Lerner, 1978).
Besides the effects under basal culture conditions,
melanin precursors have been employed also in cells
subjected to UV irradiation. Pre-incubation of the human
retinal pigment epithelial ARPE-19 cell line with 10 lM DHI
before UV-A irradiation resulted in a weak, although not
significant, protective effect (Heiduschka et al., 2007). On
the other hand, different results were obtained using
DHICA on UV-A-irradiated human keratinocytes. Supple-
mentation of low concentrations of DHICA (from 0.5 up to
2 lM) during UV-A exposure increased DNA damage as
frank single-strand breaks (SSB) in a dose-dependent
manner. Moreover, while there was no effect of the
melanin precursor on SSB in the absence of UV-A
irradiation when used at low doses (0.125–0.25 lM), the
doses of 0.5 and 1 lM significantly increased SSB with
respect to untreated cells (Kipp and Young, 1999). In our
experience on primary cultures of human keratinocytes,
the addition of DHICA (5–50 lM) to the culture medium for
24, 48, and 72 h decreases proliferation with no reduction
in keratinocyte survival. Moreover, DHICA reduces lipo-
peroxidation and apoptosis following UV-A exposure.
DHICA pre-incubation before UV-A exposure at 10 J/cm2
following a recovery of 24 h increases also cell viability
when used at 50 lM. A difference to be mentioned is that
in our experimental procedure DHICA was removed
before irradiating keratinocytes (Kovacs et al., 2012).
5SCD, the main intermediate in the metabolic pathway
of pheomelanin, showed selective toxicity to a variety of
tumor cells when added to cell medium at a concentration
of 1 mM for 1 h. 5SCD was found to be approximately 10
times more cytotoxic than L-dopa and the suggested
mechanism of action involved hydrogen peroxide produc-
tion (Fujita et al., 1980; Ito et al., 1983).
The melanin precursors 5SCD, DHI, and DHICA
together with some related metabolites are diffusible
and can be released both in body fluids and locally outside
the producing melanocytes. Cutaneous levels of 5SCD,
DHI, and DHICA have been judged to increase up to
200 lM under melanocyte-stimulating conditions such as
UV irradiation or inflammatory processes (Koch and
Chedekel, 1987). However, the precise in vitro reproduc-
tion of the local modulation of these melanin intermedi-
ates occurring under both physiological and pathological
conditions is challenging.
Natural and synthetic melanins
One possible way to evaluate the role of melanin in
preserving cell membrane lipids from UV-A-induced
peroxidation is the selective modulation of melanin
synthesis by tyrosine supplementation. In murine mela-
nocytes with different melanization (e.g., black, brown),
melanogenesis was stimulated by increasing tyrosine up
to 200 lM. Although melanin is mainly present in
melanocytes, it should be noted that it is also present in
628
a diffuse state as ‘melanin dust’, extending its pro-oxidant
or antioxidant action also to skin cells of epidermal and/or
dermal layers (Haywood et al., 2006).
A direct evaluation of the possible contribution of
natural or synthetic eumelanin or pheomelanin to the
harmful effects of UV shows several critical aspects due
to the complicated procedures for synthesis/isolation,
purification, and solubilization of these pigments. Mela-
nins should be suspended in water, stored under N2 in
the dark, and diluted in culture medium at 10 times
the desired concentrations. A number of studies have
suggested that synthetic cysteinyldopa-melanin and
dopa-melanin are suitable substitutes of natural pheomel-
anin and eumelanin. However, several aspects should be
considered to evaluate photoprotective or photo-oxidative
effects of synthetic melanins on cell cultures. Eumelanin-
induced photoprotection, for example, is directly influenced
by the effect of radiation absorption, its auto-oxidation
tendency with the production of superoxide radicals, and
its ability to act as radical scavenger.
It is unclear how these properties determine its
behavior in vivo in response to physiological levels of
radiation. Moreover, several experiments reporting higher
phototoxic effects of pheomelanin compared to eumela-
nin were performed with high-energy ionizing radiation,
and thus, the results cannot be extrapolated to UV-A and
UV-B irradiation. An additional question is, ‘In what
physical state, or states, do the melanins occur within
the melanosomes and in the keratinocytes that form
stratum granulosum and stratum corneum?’ It has been
hypothesized that melanosomes may contain soluble
melanin pigments, typically associated with melanopro-
teins, whereas it is more likely that eumelanin and
pheomelanin may form a melanin dust within keratino-
cytes. Thus, to evaluate the biological action of intramel-
anosomal melanins, soluble forms of eumelanin or
pheomelanin should be synthesized in the presence of
bovine serum albumin.
To evaluate the possible correlation between photo-
protective and/or photo-oxidative effects of cysteinyldop-
a-melanin or dopa-melanin and endogenous antioxidant
defense in mediating UV-A and UV-B cell damage, an
experimental model was set up including irradiation of
human keratinocytes in the presence of synthetic pheo-
melanin or eumelanin homologues (Briganti et al., 2005).
The pigments were suspended in PBS and diluted in cell
culture medium (15–100 lg/ml). After testing cytotoxic-
ity, 30 lg/ml was chosen as a safe concentration able to
reproduce in vitro the amount of melanins dispersed in
keratinocytes. Three experimental settings were consid-
ered: (i) short (1 h) or overnight pre-incubation of cells
with synthetic melanins and their presence during UV-A
irradiation, (ii) cell exposure to UV-A with cysteinyldopa-
or dopa-eumelanin diluted in PBS, and (iii) employing
Nunc OptiCell Culture Systems with pigment dissolved in
PBS and maintained separated from keratinocytes by a
gas-permeable polystyrene membrane. These methodol-
ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

ogies allowed to compare the biological effect of
synthetic pheomelanin or eumelanin as solution and ‘dust’
dispersed in PBS during irradiation to the pro-oxidant
action of reactive oxygen species generated by pigment’s
UV-A exposure (in particular singlet oxygen) and diffused
to cell cultures through the gas-permeable membrane.
Moreover, to distinguish the pro-oxidant or photoprotec-
tive effects of soluble melanins from those induced by
pigments in a dispersed physical state, cysteinyldopa- or
dopa-melanin was filtered through a 0.22-lm filter before
treating the cells. After UV-A exposure, the main param-
eters examined include: (i) gap junction intercellular
communication (GJIC), as index of cell integrity; (ii) cell
membrane lipids, as oxidation targets; (iii) activity of
antioxidant enzymes (catalase and glutathione peroxidase
[GSH-Px]); and amount of small molecular antioxidant
molecules (GSH and vitamin E), as index of endogenous
antioxidant system effectiveness. Synthetic pheomela-
nin, acting as a photosensitizer, was found to significantly
increase UV-A-induced GJIC inhibition, antioxidant
system imbalance, and damage of cell membrane integrity.
To summarize, use of melanin intermediates and syn-
thetic melanins on cell systems requires that several
experimental parameters are taken into account, including:
1. The specific sensitivity of the cell type utilized.
2. The light and temperature sensitivity along with the
stability of the compounds (i.e., DHICA is more stable
than DHI and leads therefore to a slower generation of
radical species in the culture medium; DHI is sensitive
to temperature increases (Maeda and Hatao, 2004;
Heiduschka et al., 2007); enzymatically prepared
dopa-melanin as well as cysteinyldopa-melanin must
either be used immediately or stored below 0°C.
Moreover, thawed samples should not be studied
after few hours at room temperature).
3. Possible reactivity in culture media during UV expo-
sure (i.e., the quick darkening of the medium when
DHI, DHICA, and related metabolites are present
during UV-A exposure due to the irreversible genera-
tion of brown pigments following their oxidation.
Consequently, keeping the compounds in the irradia-
tion medium may both increase the toxicity due to the
generation of radical species and decrease the inten-
sity of UV light reaching the cells (Maeda and Hatao,
2004; Heiduschka et al., 2007); similar cautions have
to be considered when using cysteinyldopa-melanin to
avoid photo-oxidation).
4. Conditions of the reaction mixture of synthetic mela-
nins.
5. Possible formation of aggregates when handling syn-
thetic pheomelanin or eumelanin.
Outlook and perspectives
The present paper includes different sets of recom-
mended standards and procedures that have been
ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Methods in melanin research
selected on the basis of a critical review of the literature
and experimental revision of selected methods. The main
principles and concepts underlying proposed methods
have been illustrated; detailed protocols and spectral data
of standard precursors and markers have been provided
in Appendix S1. Commercial products widely used by
pigment cell researchers have also been analyzed and
discussed. It is strongly recommended that the proposed
methods are agreed upon, and rapidly adopted by, all
laboratories working on melanogenesis and pigment cell
biology. This should eventually lead to an unprecedented
integration and synergism between research groups from
different disciplines and with various backgrounds and
expertise in the field.
Acknowledgements
This paper was carried out in the frame of the EuMelaNet Special
Interest Group and was possible thanks to the active support of the
European Society for Pigment Cell Research, the International
Federation of Pigment Cell Societies, and their officers. The contents
of the paper reflect in part presentations and discussions during
Concurrent Session 5 at International Pigment Cell Conference (IPCC
2011), Bordeaux, September 20-24, 2011 (see d’Ischia et al. Pigment
Cell Melanoma Res. 2011, 24, 782-783). The authors are grateful to
Lionel Larue, Alain Taieb, Lluis Montoliu, and Ghanem Ghanem for
encouragement and helpful discussions. MdI acknowledges financial
support by Italian MIUR, PRIN 2010-2011 (PROxi) project, for the
experimental protocols executed and verified at Naples University.
TS thanks Andrzej Zadlo for running EPR spectra of melanin samples.
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Supporting information
Additional Supporting Information may be found in the
online version of this article:
Appendix S1. Recommended protocols.
633