Effect of stacking and redox state on optical absorption spectra of melanins-comparison of theoretical and experimental results

Article abstract of DOI:10.1021/jp046710z

In this work the effect of aggregation and oxidation on the optical absorption of eumelanin oligomeric sheets is investigated by applying quantum mechanics and atomistic simulation studies to a simplified eumelanin structural model that includes 1 -3 sheets of hexameric oligomer sheets. The oligomeric hypothesis is supported by AFM characterizations of synthetic eumelanins, formed by auto-oxidation or electrochemical oxidation of dihydroxyindole (DHI). Comparison of calculated absorption spectra to experimental spectra demonstrates a red shift in absorption with oxidation and stacking of the eumelanin and validates the theoretical results.

Full text of DOI:10.1021/jp046710z

1970
J. Phys. Chem. B 2005, 109, 1970-1977
Effect of Stacking and Redox State on Optical Absorption Spectra of
Melanins-Comparison of Theoretical and Experimental Results
Klaus B. Stark*
Accelrys Incorporated, 1042 Kress Street, Houston, Texas 77020
James M. Gallas
Department Physics and Astronomy, UniVersity of Texas at San Antonio, San Antonio, Texas 78249
Gerry W. Zajac and Joseph T. Golab
BP Research Center, P.O. Box 3011, NaperVille, Illinois 60566-7011
Shirley Gidanian, Theresa McIntire, and Patrick J. Farmer
Department of Chemistry, UniVersity of California, IrVine, IrVine, California 92697
ReceiVed: July 23, 2004; In Final Form: NoVember 12, 2004
In this work the effect of aggregation and oxidation on the optical absorption of eumelanin oligomeric sheets
is investigated by applying quantum mechanics and atomistic simulation studies to a simplified eumelanin
structural model that includes 1-3 sheets of hexameric oligomer sheets. The oligomeric hypothesis is supported
by AFM characterizations of synthetic eumelanins, formed by auto-oxidation or electrochemical oxidation of
dihydroxyindole (DHI). Comparison of calculated absorption spectra to experimental spectra demonstrates a
red shift in absorption with oxidation and stacking of the eumelanin and validates the theoretical results.
Introduction
the melanin absorption spectrum may be efficient with regard
to protection against major diseases in humans.
Melanin is the biopolymer that accounts for much of the
coloration in nature.1,2 In humans it is responsible for the color
of the hair, skin, and eyes, which span a range that includes
red, yellow, brown, and black.3,4 Yet, the optical absorption
spectra corresponding to these colors have a common feature:
a broadband, structure-less curve that is a monotonically
increasing function of energy.5
This feature may reflect an efficient mechanism for photo-
protection by selective filtration of light: the wavelengths of
light are filtered in proportion to their probability to damage
tissue.6 For example, the absorption spectrum of melanin is
roughly similar to the action spectrum for retinal damage; this
damage has been associated specifically with macular degenera-
tion.7
There is also evidence that the shape of the absorption
spectrum of melanin changes with age in the retinal pigment
epithelium behind the retina, specifically becoming bleached
with loss of red-end absorption.8 In effect, after bleaching the
spectrum becomes more similar in shape to the action spectrum
for retinal damage.
The shape of the absorption spectrum of melanin also appears
to relate to skin cancer in a particular way.9 People with red
hair have a greater risk of skin cancer from exposure to sunlight.
The type of melanin in the skin of people with red or blonde
hair is called phaeomelanin and has a relatively lower optical
absorption in the red end of the visible spectrum, similar to
melanin that has been bleached or oxidized. Thus, the shape of
Theoretical work on melanin and its possible proto-molecules
is relatively sparse due to the size of the molecules involved
and the unknown nature of the overall melanin structure. Early
molecular orbital calculations on monomer units predicted
molecular properties, such as electron donor and acceptor
qualities, in accord with experimental data known for the
polymer.10 Bolivar-Martinez et al. performed ab initio and
semiempirical calculations on different monomers, indole-
quinone (IQ) and the reduced forms, semiquinone (SQ), and
hydroquinone (HQ).11 Calculations were performed for both the
neutral molecules and singly and doubly charged anions, and
the optical absorption spectra of monomeric eumelanin structures
were discussed.
In previous work we performed density functional theory
(DFT) calculations on the monomer of neutral IQ and its reduced
forms, HQ and SQ.12 Five possible dimers of the dihydroxy-
indole units were also considered and optimized. From the
optimized structures absorption spectra were calculated using
configuration interaction (CI) schemes and a semiempirical
Hamiltonian. The results agreed well with earlier theoretical
work11 as well with published experimental work.13
A similar approach was undertaken by Bochenek and
Gudowska-Nowak.14 In their work oligomers were constructed
from a random composition of the same basic monomers.
Semiempirical quantum mechanics calculations were performed
to obtain the electronic spectra for a melanin-representing model
that reproduced features of the experimental absorption spectrum
of synthetic melanins. In a separate work the effect of monomer
stacking on the electronic structure was investigated.15 Recently,
Il’ichev and Simon16 demonstrated that several tautomerization
* To whom correspondence should be addressed. E-mail:
kstark@accelrys.com.
10.1021/jp046710z CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/15/2005

Optical Absorption Spectra of Melanins
J. Phys. Chem. B, Vol. 109, No. 5, 2005 1971
equilibria for HQ and IQ should be considered in the complex
mechanism of the oxidation of IQ and that solvent polarity has
a pronounced effect on intermediate stability.
and the rational for the model is verified by AFM imaging.
Because stacking likely varies with oxidation state and thus may
be an important structural feature, extended simulations of the
optical spectra for both reduced and oxidized melanin at various
levels of stacking were performed. Quantum mechanics and
atomistic simulation studies are applied using new structural
models that include 1-3 sheets of hexameric model systems.
However, it is now well established that eumelanins form
covalently bound “proto-molecules” consisting of sheets of
indolic monomers. Beginning with extensive wide-angle X-ray
scattering (WAXS) experiments17,18 and analyses that defined
a local sheetlike structure, subsequent scanning tunneling
microscopy (STM)19-21 and atomic force microscopy (AFM)22
studies were able to identify the local structure as the funda-
mental melanin units. Such proto-molecules were described as
nanometer-sized stacks of 3.4-Å-spaced sheets. Further support
for such structures has come from matrix-assisted laser desorp-
tion ionization (MALDI) experiments, and small-angle X-ray
scattering (SAXS)23 tests indicate that melanin is likely to consist
of hydrogen-bonded aggregates of these proto-molecules.
Theoretical Calculations
Starting structures for the stacked oligomers were generated
from the monosheet structures of ref 17 obtained by the density
functional theory (DFT) optimizations. In this way, new
structural models including 1-3 sheets of oligomers were built.
The original model was also modified by allowing for partial
and full hydrolyzation of the carbonyl function. The oligomeric
nonstacked structures were optimized using the DFT program
DMol3 available from Accelrys Inc.30,31 using the same input
parameter set as in our earlier work.24 Then the stacked starting
structures were built as described above.
We therefore simulated the optical spectra of melanin using
a particular structural model that assumes a polyquinone layer
or sheet of four to six monomers with the carbonyl residing
along the perimeter of the nanometer-sized oligomers.24
A
The stacked structures included up to more than 200 atoms.
The system size would make DFT calculation very time-
consuming. Furthermore, the interactions between the sheets
are expected to be dominated by dispersion interactions, which
are not accounted for in available DFT functionals. On the other
hand, previous studies showed that the semiempirical formalism
can lead to oligomer structures with distorted chromophores.12
Therefore, on these starting structures molecular dynamics (MD)
calculations were preformed using the classical simulation
engine Discover32 and the COMPASS force field,33-37 both
available from Accelrys Inc. A script38 has been used to run
series of MD simulations in the NVT ensemble at decreasing
temperature from 800 to 300 K in steps of 50 K. The simulations
were run for 200 ps using a time step of 1 fs. The resulting
structures were reoptimized after each temperature step, and in
this way a series of structures, which were often very close in
total energy, were obtained.
On selected structures from these MD runs absorption spectra
were calculated using the pair excitation configuration interac-
tion (PECI) method39,40 and the AM1 Hamiltonian41 as imple-
mented in the semiempirical program VAMP,42 available from
Accelrys Inc. In the PECI method excited states are calculated
by including all single and double excitations in which a
complete electron pair is promoted. This is a very economical
and effective CI expansion for closed-shell molecules and is
used in VAMP for the calculation of spectra or hyperpolariz-
abilities. In the current calculations the number of orbitals, which
have been included in the PECI expansion, varied with the
number of sheets in the model (see below).
continuing problem is that quantum mechanical calculations
yield spectra with several discrete absorbance bands, whereas
the melanin spectrum is, to a large extent, continuous. However,
structural heterogeneity is inherent in the molecular structure
and molecular weights of the oligomers of natural melanins.
Thus, we proposed that the smooth optical absorption for
melanin is obtained by superposition of all the constituent
absorptions.
The structural models used in the previous work were limited
to single-sheet oligomers that ranged in size from monomer
through hexamers.12,24 In the present work the simulations
include aggregates of the oligomeric sheets as well as changes
in their oxidation states. These are important considerations for
several reasons. A variety of experimental probes point to
extensive stacking of oligomers in eumelaninsto an average
of 4-5 sheets.19,21,25 On the other hand, several investigations
support a single-sheet model for pheomelanins,26 which have
much lower absorbance in the visible region.
Changes in subunit oxidation state is relevant as the darkening
and bleaching of melanin has been correlated to oxidation/
reduction reactivity and is believed to be essential in its natural
physiological functions.27 It is therefore of great interest to
evaluate the proto-molecule model for melanin by simulating
its optical properties for oligomeric sheets in various oxidation
states and to compare such spectra with the experimental data.
We previously reported a correlation between the heights of
nanometer-sized melanin particles and the optical absorption
in the red end of the spectrum.19 Specifically, the nominal
heights of eumelanin particle was reduced from 15 to about 5
Å when the sample solutions were oxidized with hydrogen
peroxide.19,21,25 Thus, extended oxidation causes a destacking
which decreases the relative optical absorption in the red part
of the optical spectrum of melanin.
In previous investigations of the effect of oligomer size on
the optical spectra it was found that the optical absorption for
longer wavelengths increased with oligomer size.24 To focus
this work on the effects of stacking and redox state on the
simulated optical spectra the oligomer size is held constant at
hexamers, which was suggested by previous WAXS experiments
as the smallest common oligomeric size in natural melanins17,18
and was the largest system studied in ref 24.
Natural melanins are highly heterogeneous mixtures contain-
ing dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic
acid (DHICA), and other subunits, protein substituents, and
absorbed metals. However, in this work we will consider only
the simplest components that define the main characteristics of
eumelanin, derived from DHI subunits.28 Upon electrochemical
oxidation DHI polymerizes on the surface of electrodes, which
facilitates its handling and chemical characterization. DHI-
melanin’s electrochemical behavior and speciation has been
described in the literature.28,29 The results of theoretical calcula-
tions are compared to experimental spectra of DHI-melanin,
Experimental Section
Materials and Methods. The reagent 5,6-diacetoxyindole
(DAI) was obtained from TCI America, Portland, OR. Water
used in all experiments was 18 MΩ/cm3 Nanopure water.
HPLC-grade isopropyl alcohol was purchased from Merck. All
other chemicals were reagent-grade quality. Absorbance spectra
were obtained on a Hewlett-Packard 8453 UV-vis spectrometer

1972 J. Phys. Chem. B, Vol. 109, No. 5, 2005
Stark et al.
Figure 1. DFT-optimized hexameric DHI melanin structures: (a) oxidized form; (b) half-reduced form; (c) fully-reduced form. Carbon atoms are
displayed in gray, oxygen atoms in red, nitrogen atoms in blue, and hydrogen atoms in white.
or a Cary 500 UV-vis-NIR spectrophotometer. Electrochemi-
cal measurements were conducted with a BAS-100B-W elec-
trochemical analyzer using a three-electrode voltammetric cell
with a platinum counter electrode and Ag/AgCl as reference
electrode.
carried out by either bulk electrolysis or cyclic voltammetry of
a DHI solution using freshly cleaved HOPG as the working
electrode. A DHI solution was prepared by hydrolyzing DAI
in situ: DAI (0.030 mmol, 6.0 mg) was dissolved in 12.5 mL
of degassed ethanol to which 2 equiv of 0.1 M NaOH (0.55
mL), needed to hydrolyze the acetoxy groups, was added. This
solution was allowed to stand for 1 h and then diluted to 25
mL with pH 7.0, 0.05 M phosphate buffer. Polymerization of
DHI on HOPG surface was achieved by either bulk electrolysis
at 20 mV for 6 s or cyclic voltammetry for three cycles at a
sweep rate of 500 mV s-1 for potentials varying between -0.40
and 0.35 V vs Ag/AgCl. The plates were then rinsed with
deionized water and allowed to dry overnight.
AFM images were collected on a Park Scientific AutoProbe
microscope operating in noncontact mode, equipped with
NCAFM probe head. In a typical procedure a piezoelectric
scanner with a range to 10 µm was used for all images. The
scanner was calibrated in the xy directions using a 1.0-µm
grating and in the z direction using conventional height
standards. The tips used were V-shaped silicon 2 µm cantilevers
(Ultralevers, model ULNC-AUNM, Park Scientific Instruments).
The oscillation amplitude and z direction set point were adjusted
to avoid tip-sample contact according to operating procedure
for noncontact mode imaging. All measurements were per-
formed in air at ambient pressure and humidity. Images were
stored as 256 × 256 point arrays and analyzed using AutoProbe
image processing software supplied by Park Scientific Instru-
ments.
Electrochemical DHI Melanin Deposition and Charac-
terization. Preparations of DHI melanin were performed in a
glovebox under a nitrogen atmosphere. A sample of DAI (0.030
mmol, 6.0 mg) was dissolved in 12.5 mL of degassed ethanol
to which 2 equiv of 0.1 M NaOH (0.55 mL) was added. This
solution was allowed to stand for 1 h and then diluted to 25
mL with 0.05 M, pH 7.0 phosphate buffer. Thick films of DHI
melanin were deposited on the electrode surface using cyclic
voltammetric scans varying between -0.40 and 0.35 V vs Ag/
AgCl for 15 cycles at a sweep rate of 50 mV s-1
.
The spectroelectrochemical methodology is described in detail
elsewhere;29 briefly, a thick melanin film was deposited on ITO
plate and placed in a three-neck, anaerobic UV-vis cell
containing pH 7.0 buffer solution. After obtaining the absor-
bance spectrum of the film without applied potential as a
reference the ITO electrode was connected to the electrochemi-
cal analyzer as working electrode, using a platinum wire as
counter electrode and an Ag/AgCl reference electrode. Variable
potentials were applied to the film, and the absorbance spectrum
of the film was obtained. Subtraction of the reference spectrum
from these spectra provided the background-subtracted spectra.
Preparation of DHI Melanin Samples for AFM Examina-
tion. To prepare auto-oxidized DHI melanin, stock solutions
of synthetic DHI-melanin were prepared by hydrolysis of 120
mg (0.5145 mmol) of 5,6-diacetoxyindole (DAI) with 20%
molar excess of KOH (1.28 mmol) in 250 mL of deionized
water. The reaction mixture was stirred rapidly in air, allowing
auto-oxidation of the hydrolysis product 5,6-dihydroxyindole
(DHI). The stock solution was then degassed and stored under
a flow of purified N2 presaturated with water. The stock solution
was diluted 100-fold in 1:4 2-propanol/water, and 10 µL of the
resulting melanin solution was deposited onto a freshly cleaved
highly oriented pyrolytic graphite (HOPG) surface, previously
mounted on stainless steel pegs by double-stick tabs. The
samples were allowed to dry overnight before analysis by AFM.
Electrochemical deposition of DHI melanin on HOPG was
Results
Theoretical Calculations on Eumelanin. In previous work
an oligomeric model of eumelanin was developed using the fully
oxidized form of melanin.24 Here, this chemical model is
extended by including half-reduced and fully-reduced proto-
molecules as well as the oxidized form. These were built starting
from the optimized oxidized form obtained earlier, and the
nonstacked new oligomers were optimized using similar DFT
procedures.24
Shown in Figure 1 are the resulting energy-optimized
hexamers for the three oxidation states. For the oxidized form

Optical Absorption Spectra of Melanins
J. Phys. Chem. B, Vol. 109, No. 5, 2005 1973
the oxidized form. The predicted absorption in the red part of
the spectrum is also lower in the reduced form. The absorption
spectrum of the half-reduced form exhibits intermediate behav-
ior, with absorbance bands over a broad wavelength range.
The large number of possible hydrogen-bonding interactions
precluded simple energy minimization of layered structures in
the reduced and half-reduced forms; therefore, the molecular
dynamics quenching technique was applied to generate a
manifold of low-energy structures for the stacked oligomers.
Only intrasheet hydrogen bonds are expected for the oxidized
structure, and consequently, the quenching procedure led to well-
defined low-energy structures in which a stacked structure is
built with the individual sheets somewhat shifted against each
other. An example for such a structure is shown in Figure 4a.
Optical spectra were calculated for the oxidized hexamer stacked
in 1-3 sheets, the results being displayed in Figure 5. In these
calculations the number of orbitals used for the CI expansion
has been increased with the number of sheets N, so as to
maintain the sampling of energy levels at 7 eV around the
HOMO-LUMO gap. This lead to a CI expansion including 8,
12, and 16 orbitals for N ) 1, 2, and 3,respectively. Overall
the spectra become more red shifted and smooth with increasing
number of sheets N. A qualitatively very similar picture (not
shown in Figure 5) was obtained with a constant CI expansion
into 10 orbitals for all N. These spectra, albeit different in details,
showed the same trend in red shift with increasing number of
sheets.
Figure 2. Frontier orbitals of the half-reduced hexamer of Figure 1b
calculated with VAMP: (a) HOMO, (b) LUMO. Positive orbital lobes
displayed in blue; negative orbital lobes displayed in yellow.
The variable CI expansion used to generate absorption spectra
in Figure 5 is certainly justified when comparing the effect of
stacking because it reflects the clustering of more energy levels
at the same energy interval with increasing N. However, we
can only be concerned with qualitative effects here since, first,
our model for eumelanin is only approximate and still incom-
plete and, second, with increasing size of the model estimating
the convergence of the CI expansion becomes more and more
difficult due to the increased clustering of orbitals mentioned
above. Therefore, for the absorption spectra in the other figures
of this work the CI expansion is reduced to include only 10
orbitals, which reduces the computational effort while still
reflecting the same general trends.
Figure 3. Lorentzian broadened calculated absorption spectra for
single-sheet hexamers of the oxidized, half-reduced, and fully-reduced
melanin proto-molecules.
Examples of low-energy structures of hexamers of the half-
reduced and reduced form obtained by the quenching procedure
are shown in Figure 4b and c, respectively. Several structures
exhibited very similar total energies, and typically 4-8 struc-
tures were found in a narrow energy range of 10-20 kcal/mol.
Therefore, it is assumed that more than one structure contributes
to the absorption spectrum of the half-reduced and reduced form.
Calculated absorption spectra for selected low-energy structures
of the half-reduced and reduced forms are shown in Figure 6.
To generate these spectra the number of sheets was held constant
at N ) 3, following the model generated by WAXS data on
enzymatically derived melanin,17,18 and also keeping the cal-
culation time reasonable. In addition, the CI expansion was
reduced to include only 10 orbitals (see discussion of Figure 5
above).
(Figure 1a) two hydrogen-bond interactions are found which
include a strong N-H interaction (r ) 1.69 Å) and a weak O-H
interaction (r ) 2.19 Å). Naturally, more hydrogen bonds can
form for the half-reduced form (Figure 1b) and the fully-reduced
form (Figure 1c) between adjacent hydroxyl groups or between
the hydrogen bond of a hydroxyl group and the adjacent
carbonyl oxygen. For the half- and fully-reduced structures it
is expected that hydrogen bonds will form both intra- and
intersheet and that this manifold of possible hydrogen-networked
isomers will affect the absorption spectra. This effect will be
less pronounced for the oxidized form (vide infra).
Frontier orbitals (calculated with VAMP) are displayed for
the half-reduced form in Figure 2, which shows the highest
occupied molecular orbital (HOMO, Figure 2a) and the lowest
occupied molecular orbitals (LUMO, Figure 2b). The HOMO
has π-character and is extensively distributed around the entire
molecular system, whereas the LUMO (π*) is more localized
to the interior portions of the system and excludes for the most
part the oxygen-containing groups.
Synthetic Eumelanin. Melanin samples from DHI were
prepared by both auto-oxidation and electrochemical deposition.
Cyclic voltammograms of both forms is similar in that a sharp
irreversible oxidation wave is observed with a peak current of
ca. +100 mV vs Ag/AgCl, with a much weaker broad reduction
current seen in the opposite scan direction (Supporting Informa-
tion, Figure S1).
Calculated absorption spectra for the single-sheet structures
are shown in Figure 3. The raw theoretical spectra have been
Lorentzian broadened as described previously.12 The absorption
maximum of the reduced form is red shifted as compared to
Figure 7 shows an AFM image and the particle size
distribution of submonolayer eumelanin deposited by electro-

1974 J. Phys. Chem. B, Vol. 109, No. 5, 2005
Stark et al.
Figure 4. Low-energy structures for the three sheets of hexamers of the (a) oxidized, (b) half-reduced, and (c) reduced forms. For clarity, the
structures are displayed in stick representation. Hydrogen bonds are displayed as light blue dashed lines.
Figure 5. Lorentzian broadened and normalized calculated absorption
spectra for hexamers of the oxidized form. Displayed are the spectra
of the nonstacked form (1 sheet) and stacked forms with 2 and 3 sheets
(for more details see text).
chemical oxidation of DHI on HOPG. Analysis gives the
average particle height as 27.93 Å (standard deviation of 8.19
in a sample of 207 particles measured). The particles show a
tendency to align at step edges of HOPG. By comparison, AFM
imaging of colloidal melanin prepared from DHI auto-oxidation
identified particles ranging from 7 to 22 Å in height, with few
larger particles up to 35 Å seen (Supporting Information, S2).
Statistical analysis gave the average particle height as 13.83 Å
(standard deviation of 4.51 in a sample of 204 particles
measured). Imaging of the micrometer-thick DHI depositions
Figure 6. Absorption spectra of low-energy structures of hexamers
of the half-reduced (upper panel) and reduced forms (lower panel).
The number of sheets for all structures considered is held constant at
N ) 3.
is also indicative of granularity of roughly the same scale, as
illustrated in Figure 8. Note that due to the much larger size of
interconversion of stable species. Under anaerobic conditions
the AFM probe tip, the lateral dimensions in these images do
this absorbance change is reversible and can be repeated several
not correspond to the lateral sizes of the particles themselves;
times. The resting potentials of an as-made film are generally
in the range of 0-50 mV, i.e., within the chemically reversible
only the height relative to the HOPG substrate can be measured
accurately.
region. At potentials in the range from +50 to +300 mV the
characteristic increase at 500 nm continues but the reversibility
is lost, indicating a change in the chemical composition. Thick
DHI melanin films treated with hydrogen peroxide display
similar absorbance changes, Supporting Information, S3, but
the isosbestic point at ca. 417 nm is lost almost immediately.
The absorbance spectra obtained by electrolysis of a thick
DHI melanin film at various potentials is shown in Figure 9.
As the potential of the film is altered from its fully-reduced
state at -300 mV, the absorbance increases above 400 nm with
increasing potential. Between -300 and 50 mV an isosbestic
point at ca. 417 nm is maintained, which denotes a simple

Optical Absorption Spectra of Melanins
J. Phys. Chem. B, Vol. 109, No. 5, 2005 1975
for its smoothness and lack of any apparent bands throughout
the visible, near-IR, and near-UV region of wavelengths?
In previous work24 we simulated spectra for tetrameric,
pentameric, and hexameric DHI oligomers. The most striking
result was that the peaks of the various oligomers shift to the
red part of the spectrum with increasing oligomer size. Thus, it
is possible that with sufficient heterogeneity a superposition of
such spectra one could replicate the smooth melanin optical
absorption. Additional heterogeneity could be due to other
structural variants of a given oligomer size, for example, those
resulting from substituents such as carboxylate functionalities
at the oligomer periphery which are not considered here.
Structural Models of Eumelanin. AFM images of DHI
melanin all show nanoscale particles; the colloidal samples have
average particle heights of 13.8 Å, which match well with
previous reports on synthetic melanins.19,21 The average height
of electrochemically deposited particles is 27.9 Å (std 8.2 Å),
which corresponds to 6-7 stacked sheets and roughly agrees
with the ∼2-nm-sized molecular structures calculated in this
study. Formation of bigger particles by electrochemical deposi-
tion in comparison with auto-oxidation is probably due to the
difference in polymerization conditions and nucleation on HOPG
surface. Complete size agreement is not to be expected given
that SAXS studies have indicated various states of aggregation
of stacked sheets exist in dilute solutions.25
Changes in Absorbance with Oxidation. For the nonstacked
oligomers (Figure 3) the absorption in the UV part of the
spectrum of the reduced form is red shifted compared to the
oxidized form accompanied by a reduction of the absorption in
the infrared part of the spectrum. The absorbance spectrum of
the nonstacked half-reduced form exhibits intermediate behavior
and has peaks over the entire wavelength range.
Figure 7. (Top) AFM image of DHI melanin prepared by cyclic
voltammetry on HOPG. (Bottom) Histogram of percent population vs
height for DHI melanin particles. Statistical analysis of 10 separate
regions, N ) 207.
Stacking increases the absorption in the red-end part of the
spectrum and smoothes the absorption spectrum (Figure 5). For
the half- and fully-reduced forms a variety of structures are
expected to contribute to the absorption spectrum due a manifold
of possible hydrogen networks that can form during the stacking
procedure. For the half-reduced (Figure 6, top) form the stacked
model exhibits less absorption in the UV spectral range
accompanied by an increase of absorption on the red end of
the spectrum. For the fully-reduced form (Figure 6, bottom)
stacking narrows the spectral range in which the reduced form
absorbs to a region of 300-500 nm.
Figure 8. AFM image of thick DHI melanin film prepared by cyclic
voltammetry on HOPG.
The effect of oxidation on the absorbance of DHI-melanin
was demonstrated by spectroelectrochemical measurements on
a thick film on transparent ITO electrodes, which allows fine
control over the redox state of the sample. The visible spectra
of colloidal melanins initially darken when oxidized, which may
be a result of both the oxidation state of the catecholic subunits
as well as increased scattering due to aggregation. This problem
is avoided using DHI-melanin film electrodes,
The calculated optical absorption spectra generally indicate
a trend, as shown in Figures 3, 5, and 6, of a stronger absorption
in the red end of the spectrum with higher oxidation state of
the melanin. Thus, the calculations agree in general terms with
the experimental observations. However, this does not prove
that this particular molecular structure as utilized for these
calculations is unique and that other structures would not
likewise red shift with higher oxidation state. However, it is
important to note that the spectral region affected by the
electrochemical oxidation of melanin is very similar to those
theoretically calculated for these structures (300-700 nm). We
take this to indicate that the molecular structures most likely
Figure 9. Changes in the optical absorption spectra of a thick DHI-
melanin film on ITO during electrochemical oxidation from -300 mV;
spectra taken after redox equilibration at (a) -300, (b) -100, (c) -50,
(d) 0, (e) 50, (f) 100, (g) 200, (h) 300, and (i) 400 mV vs Ag/AgCl.
Discussion
In our initial simulations of the optical spectra of melanin it
was surprising to see discrete optical bands associated with the
nanometer-sized single oligomer obtained by density functional
theory calculations. How could such discrete optical bands lead
to the experimental melanin absorption spectrum, well known

1976 J. Phys. Chem. B, Vol. 109, No. 5, 2005
Stark et al.
involved are of similar size and degree of conjugation to those
considered in this study.
the melanin. Although this does not prove that the particular
models used for the theoretical calculations are unique and that
other structures would not also red shift with higher oxidation
state, the agreement is an indicator that the molecular structures
most likely involved in eumelanins are of similar size and degree
of conjugation to those used in this work.
Stacking significantly red shifts the absorbance spectra of the
hexameric eumelanin model system and likely plays a major
role in the broad absorbance to the near-IR that is typical of
eumelanins. Destacking, as engendered by, e.g., ring-cleavage
of the DHI monomers within an oligomer sheet, can be the cause
for the bleaching of the near-IR absorbance upon oxidation.
Effect of Stacking. The effect of stacking can be discerned
by comparing Figures 3 and 6. In the half-reduced hexamer
(Figure 6, top) less absorption in the UV part of the spectrum
is retained. Most of the absorption occurs in the range from
400 to 600 nm. For the fully-reduced form the effect of stacking
is not as pronounced. However, stacking seems to narrow the
spectral range in which the reduced form absorbs to a region
of 300-500 nm, whereas the nonstacked form (Figure 3) also
had substantial absorption below 300 nm.
The overall effect of stacking is similar to that of oxidation,
red shifting the absorbance spectra of the hexameric eumelanin
model systems. This may explain the broad absorbance of
natural melanins that extends to the near-IR; the lack of such
stacking might explain the spectra of pheomelanin, as found in
the skin of people with red or blonde hair. Pheomelanin contains
a high proportion of cysteine incorporated during the oxidative
polymerization of L-dopa, and X-ray scattering does not indicate
the average distance between sheets of 3.4 Å as in eumelanin.26
Likewise, pheomelanin’s absorbance drops off dramatically past
500 nm and more closely resembles that of highly oxidized DHI
melanins.
Such destacking effects are likely the cause of the significant
bleaching at high wavelengths seen in Figure 9. Within a specific
range the oxidation of DHI melanin is chemically reversible,
but beyond 100 mV the spectral changes are irreversible,
indicating oxidative degradation. Overall, the oxidation of DHI
melanin by hydrogen peroxide engenders similar spectral
changes as observed during electrochemical oxidations, but there
is an immediate loss of the isosbestic point indicating oxidative
degradation of the eumelanin structures. Peroxidation of mela-
nins has been proposed to induce ring opening of the DHI
catechols forming bis-carboxylate derivatives;27 such ring-
opening decomposition products have been identified by mass
spectrometry in both native and synthetic melanins.43-46 Ring-
opening decomposition of a DHI oligomer would decrease its
effective conjugation and likewise decrease its propensity to
stack due to increased sterics and solubility.
Supporting Information Available: Three supplemental
figures. This material is available free of charge via the Internet
at http://pubs.acs.org.
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