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Morphology-sensitive Raman modes of the malaria pigment hemozoin†

Torsten Frosch,*ab Sasa Koncarevic,c Katja Beckerc and J€urgen Poppab
Received 3rd December 2008, Accepted 2nd April 2009
First published as an Advance Article on the web 9th April 2009
DOI: 10.1039/b821705j

Resonance Raman spectroscopy was applied for investigating the malaria pigment hemozoin, which is
an important target structure of antimalarial drugs. Morphology-sensitive low wavenumber modes of
hemozoin were selectively enhanced with help of excitation wavelengths at l ¼ 633 nm and l ¼ 647 nm.
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The assignment of the most prominent bands in the Raman spectra at 343 cm1 and 368 cm1 was
assisted by DFT calculations of the hemozoin dimer. The mode at 343 cm1 in the Raman spectrum
of hemozoin is strongly enhanced with lexc. ¼ 647 nm and is represented by a combined, symmetric
doming mode of the two hematin units in the hemozoin dimer. The enhancement of this vibration is
stronger in the resonance Raman spectrum of hemozoin compared with less crystalline b-hematin. The
selective resonance enhancement of the morphology-sensitive Raman modes of hemozoin is caused by
absorption bands in the UV-VIS-NIR spectrum. This absorption spectrum of the crystalline malaria
pigment hemozoin shows a strong band at 655 nm. Another broad absorption band at 870 nm is the
reason for the strong relative resonance enhancement of the mode at 1372 cm1 in the Raman spectrum
of crystalline hemozoin with lexc. ¼ 830 nm. In conclusion, resonance Raman micro-spectroscopy with
lexc. ¼ 647 nm was shown to have great potential as an analytical tool to probe the morphology of
hematin samples.

Introduction To study this aspect, Raman spectroscopy14–16 has unique

Tropical malaria represents a major obstacle to human health
and welfare in sub-Saharan Africa, where every 30 seconds
a child younger than 5 years is killed by the disease.1–3 The
multiplication of the erythrocytic stages of the malaria parasite
Plasmodium falciparum and their release into the blood stream
are the major causes of the symptoms observed. During its
intraerythrocytic development the parasite consumes up to 80%
of the human host cells’ hemoglobin in its food vacuole. As
a result of this process, free, toxic heme is released and subse-
quently detoxified via bio-crystallization to the malaria pigment
hemozoin. The non-toxic needle-shaped hemozoin crystals are
stored as inert deposits.
Antimalarial key-drugs such as chloroquine4,5 interfere with
this heme detoxification process,6–11 most probably by interac-
tion with hemozoin.12,13 Chloroquine and other quinoline anti-
malarials are likely to block the small active growing face of the
hemozoin crystals and thus act as crystal growth inhibitors.12,13
Despite the tremendous public health impact of malaria and the
urgent need for improved antimalarial strategies little is known
about the molecular interaction of chloroquine and other anti-
malarial drugs with the important intracellular target hemozoin.
a
Institut f€
ur Physikalische Chemie, Friedrich-Schiller-Universit€
at Jena,
Helmholtzweg 4, D-07743 Jena, Germany. E-mail: torsten.frosch@gmx.
de; Tel: +49 3641 948351
b
Institut f€
ur Photonische Technologien e.V., Albert-Einstein-Straße 9,
D-07745 Jena, Germany
c
Interdisziplin€
ares Forschungszentrum, Universit€
at Giessen, Heinrich-Buff-
Ring 26-32, D-35392 Giessen, Germany
† This paper is part of an Analyst themed issue on Optical Diagnosis. The
issue includes work which was presented at SPEC 2008 Shedding Light
on Disease: Optical Diagnosis for the New Millennium, which was held
in São Jose dos Campos, São Paulo, Brazil, October 25–29, 2008.
potential. It can be employed for a fast, label-free and non-
invasive investigation of drug–target-interactions on a molecular
level.17,18 Due to its high sensitivity and selectivity, resonance
Raman spectroscopy is especially suited for analysing biological
systems.19,20 The combination of a Raman setup with a conven-
tional light microscope allows for a resonance Raman spectro-
scopic investigation of living cells with a spatial resolution of
about 0.5 mm.21,22 In a series of experiments, it was shown that
the Raman spectra of the antimalarial drugs chloroquine,23
mefloquine24 and quinine25 can be monitored in therapeutically
meaningful concentrations with the help of the UV resonance
enhancement of the Raman spectra. Also the influence of pH23
and aqueous environment25 on the spectra is well understood and
even small amounts of antiparasitic compounds like quinine
and dioncophylline A could be localized in cinchona bark24 and
Triphyophyllum peltatum.26,27 Density functional theory (DFT)
calculations of the Raman spectra were shown to be very helpful
for a thorough assignment and interpretation of the experimental
findings.22–28 Furthermore, the malaria pigment hemozoin could
be localized in infected red blood cells using resonance Raman
micro-spectroscopy.22,29–31 Single erythrocytes were scanned with
a spatial resolution of 0.5 mm, and Raman images of the hemo-
zoin distribution in P. falciparum were built on the basis of the
spectral array.22 The binding affinity of chloroquine to hematin
in solution was investigated in vitro.17,18 Polarization-resolved
resonance Raman experiments elucidated the detection of
a change of the depolarization ratio of the n19 mode of hematin
(Fe(III)ProtoporphyrinIX-OH) at 1569 cm1, which is based on
the interaction with chloroquine.17 This inverse polarized n19
mode is very sensitive for symmetry lowering of the hematin
molecules due to the attachment of quinolines. Small wave-
number shifts were found employing Raman difference

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spectroscopy.18 These results further prove the hypothesis of
a weak interaction between the drugs and the targets in solution
and may support the idea that free heme is involved in the
primary drug binding process.10,11
Taken together, Raman spectroscopy was shown to be
a powerful method for investigating the mode of action of anti-
malarial drugs.17,18 Further experiments will help to understand
the bio-crystallization of the malaria pigment and the drug–
target-interaction in more detail. This paper deals with a struc-
tural study on hemozoin. Resonance Raman micro-spectroscopy
was applied to selectively enhance morphology-sensitive Raman
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modes of different hematin samples. The results further support
the great potential of resonance Raman micro-spectroscopy as
an analytical tool in malaria research.
Materials and methods
Extraction of hemozoin
Blood stages of the P. falciparum strain Dd2 (chloroquine-
resistant) were maintained in culture using a modified protocol of
Trager and Jensen.32,33 RPMI medium 1640 supplemented with
NaHCO3 and HEPES, pH 7.4, 20 mg/ml gentamicin sulfate, 2
mM glutamine, 200 mM hypoxanthine, 0.2% Albumax II, and
4.1% of human serum was used for cultivation. Parasites were
isolated by lysing red blood cells for 10 min at 37  C in 20
volumes of saponin containing buffer (7 mM K2HPO4, 1 mM
NaH2PO4, 11 mM NaHCO3, 58 mM KCl, 56 mM NaCl, 1 mM
MgCl2, 14 mM glucose, 0.02 mM saponin, pH 7.5). The pellet
obtained by centrifugation (1500 g, 5 min, 4  C) was washed
three times, and subsequently the P. falciparum cells were
disrupted by three cycles of freezing in liquid N2 and thawing.
The supernatant after centrifugation at 13 000 g for 30 min was
discarded. The obtained brownish pellet was lyophilized and
hemozoin was extracted according to ref. 34. The dry pellet was
subsequently extracted with chloroform–methanol (2 : 1 v/v) and
chloroform–methanol–water (10 : 10 : 3 v/v). After drying, the
residue was resuspended in 100 mM Tris-HCl, 1 mM CaCl2, pH
7.5, and digested with pronase to remove proteins and protein-
linked GPIs. The insoluble residue was extracted with 50 mM
sodium phosphate, pH 7.2, 4 M guanidine hydrochloride, 0.5%
Triton X-100 and stirred overnight at 4  C to remove nucleic
acids. The residue (insoluble pigment, hemozoin) was recovered
by centrifugation and washed three times with water, once with
80% 1-propanol, and dried.
Synthesis of b-hematin
Chemicals were purchased from Sigma-Aldrich and Merck.
b-Hematin was synthesized according to the acid-catalyzed
dehydration of hematin (Fe(III)ProtoporphyrinIX-OH).35,36 A
solution of 0.109 g hemin (Fe(III)ProtoporphyrinIX-Cl) in 25 ml
0.1 M NaOH was stirred for 30 min under reflux. Propionic acid
was added until the pH reached 4.1. During the addition of the
first drops of the acid the precipitation of a dark solid could be
observed. The suspension was then stirred for 18 h at 70  C under
reflux. After cooling at ambient temperature the reaction mixture
was filtered through a nylon filter (Millipore, 22 mm), the solid
was washed alternating with methanol and water and finally with
0.1 M NaHCO3 solution. The procedure was finished by washing
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with water until the filtrate became neutral. The black solid was
dried over phosphorus pentoxide.
Spectroscopy
The non-resonant Raman spectra of the samples were recorded
with a Bruker FT Raman spectrometer (RFS 100/S) at the
macroscopic mode with a spectral resolution of 2 cm1. A
Nd:YAG laser operating at its fundamental wavelength of 1064
nm with an estimated laser power at the samples of approx. 100
mW was used as the excitation source. The Raman scattered light
was collected by means of a liquid nitrogen cooled Ge detector.
The resonance Raman spectra were acquired with a Raman
setup (HR LabRam, Horiba/Jobin-Yvon) equipped with an
Olympus IX71 microscope, a video camera and liquid nitrogen
cooled CCD detector. An Olympus 20/0.5 objective focused the
laser light on the samples. As excitation wavelengths the 568 nm,
647 nm and 676 nm lines of a krypton ion laser (Coherent Innova
300C) as well as 532 nm line of a frequency doubled Nd:YAG
laser (Coherent Compass) and the 633 nm line of a HeNe laser
were used. Validation of the wavenumber axis was performed
between the measurements using the Raman signals from TiO2
(anatase).
UV-VIS-NIR absorption spectra were acquired with a Cary
5000 spectrophotometer (Varian). Solid samples were suspended
in Nujol oil and a homogenous layer was smeared on CaF2
substrates.
Density functional theory calculation
DFT calculations were performed with Gaussian 0337 applying
the model chemistry BP86/SDD.38,39 The geometry of the
hemozoin dimer was optimized starting from an X-ray structure
derived by Pagola et al.12 Subsequently the Raman intensities
were calculated from the Raman scattering activities.14
Results and discussion
As explained in the Introduction – the malaria pigment hemo-
zoin is a major target for antimalarial drugs.10–13,22,40–45 Therefore
the investigation of the structure and the crystallization process
of hemozoin are of major importance. It was shown that
hemozoin (Fig. 1) can be generated in a purely chemical process42
and that it consists of hematin (Fe(III)ProtoporphyrinIX-OH)
units which are linked to dimers by reciprocal Fe1–O41 iron–
carboxylate bonds between the central iron atoms and the
propionic acid side chains (Fig. 1).46 These dimers are further
connected by hydrogen bonds and build up a regular network –
the triclinic hemozoin crystal.12 This in vitro synthesized
substance is called b-hematin. The synthesis of b-hematin opens
up the possibility for a study of drug–target-interactions in
a controlled chemical environment. It was shown that b-hematin
is the chemical,46 IR spectroscopic46 and crystallographic47
analogue of the malaria pigment. There are two established ways
for a synthesis of b-hematin. The dehydration of hema-
tin(Fe(III)ProtoporphyrinIX-OH)35,36 leads to a more amorphous
structure of b-hematin compared with the natural hemozoin,
while a very crystalline morphology can be synthesized following
the anhydrous dehydrohalogenation of hemin (Fe(III)Protopor-
phyrinIX-Cl).48,49 The resonance Raman spectra of synthesized
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Fig. 1 Molecular structure of the dimeric unit cell of the malaria
pigment hemozoin. The unit cell of hemozoin consists of a dimer of
hematins.12 The two hematin molecules themselves are bonded by
reciprocal Fe1–O41 iron–carboxylate linkages.12,45 The dimers are further
connected via hydrogen bonds (between O36 and O37) and build up
a triclinic crystal.12
b-hematin22,44 and extracted hemozoin22 were recently investi-
gated and show a variety of distinct enhancement patterns for
different excitation wavelengths. The comparison of a broad
range of Raman spectra of hemozoin and of b-hematin proved
that b-hematin is the synthetic analogue of hemozoin.22
However, the non-resonant Raman spectra with lexc. ¼ 1064 nm
revealed some tiny differences in the low wavenumber spectral
region of hemozoin compared to b-hematin samples with
different sample morphology.22 These Raman modes were
studied more thoroughly in this contribution with the help of
selective resonance enhancements.
The malaria pigment hemozoin was extracted from P. falci-
parum.22,34 The Raman spectra with excitation wavelengths lexc.
¼ 1064 nm, 830 nm, 676 nm, 647 nm, 633 nm, 568 nm, and 532
nm are shown in Fig. 2 and some prominent wavenumber values
are given in the spectrum with lexc. ¼ 647 nm. The comparison of
the different resonance Raman spectra shows some dramatic
selective mode enhancements – most prominently the enhance-
ment of the band at 1372 cm1 for excitation wavelengths 568 nm
and 830 nm (Fig. 2). This important Raman mode is known as p
density marker band19 and was assigned to a vibration localized
at the four pyrroles which overlap in the region where the two
hematins are linked with each other (Fig. 1).22 It is obvious that
the relative resonance Raman enhancement of such a vibration is
influenced by the closely packed porphyrins in the three-dimen-
sional structure of hemozoin. This effect is an aggregation
enhancement.44,50
These resonance effects in the Raman spectra of the aggre-
gated hemozoin originate from electronic transitions. Significant
bands are therefore expected in the UV-VIS-NIR absorption
spectra. The UV-VIS-NIR absorption spectrum of hemozoin is
presented in Fig. 3A alongside with the spectrum of b-hematin in
Fig. 3B. First of all a dramatic decrease of the Soret band
absorption is seen due to the sample aggregation (slightly
stronger for the more crystalline hemozoin in Fig. 3A).
Furthermore an absorption band at approx. 870 nm is observed
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Fig. 2 Raman spectra of the extracted malaria pigment hemozoin taken
with excitation wavelengths: lexc. ¼ 1064 nm, 830 nm, 676 nm, 647 nm,
633 nm, 568 nm, and 532 nm. Some prominent wavenumber values of
hemozoin are given in the resonance Raman spectrum with lexc. ¼ 647
nm. Different relative resonance enhancements are seen for the various
excitation wavelengths.
in both spectra of hemozoin (Fig. 3A) and b-hematin (Fig. 3B).
This absorption explains the dominant enhancement of the
Raman band at 1372 cm1 for excitation with lexc. ¼ 830 nm
(Fig. 2). While a recent DFT calculation22 already gave some
explanations for this enhancement (due to the fact that the mode
is localized at the four overlapping pyrroles around the propio-
nate linkage of the hemozoin dimer), the absorption spectra
prove the presence of a broad electronic transition. A closer
analysis might even elucidate a more pronounced absorption
band of hemozoin (Fig. 3A) compared with the less crystalline b-
hematin sample (Fig. 3B).
Some recent results derived with non-resonant Raman spec-
troscopy22 lead the interest to a comparison of the resonance
Raman spectra of hemozoin (Fig. 4A1, 4B1, and 4C1) with b-
hematin in a more amorphous structure. Therefore b-hematin
was synthesized following the dehydration of hematin (Fe(III)-
ProtoporphyrinIX-OH).34,35 The IR spectrum of the sample
shows the significant bands at 1712 cm1, 1664 cm1, and 1211
cm1 (results are not shown) and the resonance Raman spectra
are illustrated in Fig. 4A2, 4B2, and 4C2. The overall agreement
of the spectra is good and further evidence for the analogy of
hemozoin and b-hematin. However, a closer analysis of the low
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Fig. 3 Absorption spectra of hemozoin (A) and b-hematin (B). The
different absorption bands give rise to various relative resonance
enhancements in the Raman spectra (Fig. 2 and 4).
wavenumber modes at 343 cm1 and 368 cm1 elucidates
a significantly stronger resonance enhancement of these bands in
the Raman spectra of hemozoin (Fig. 4A1 and 4B1) with exci-
tation wavelengths of 633 nm and 647 nm compared with the
spectra of b-hematin (Fig. 4A2 and 4B2). The most pronounced
differences are observed for the strongly enhanced mode at
Fig. 4 Comparison of the resonance Raman spectra of hemozoin (A1, B1, and C1), b-hematin (A2, B2, and C2), hematin (Fe(III)ProtoporphrinIX-OH)
(A3, B3, and C3), and hemin (Teichmann crystals) (A4, B4, and C4) with laser excitation wavelengths A: lexc. ¼ 633 nm, B: lexc. ¼ 647 nm, and C: lexc. ¼
676 nm. Some prominent wavenumber values are given in the resonance Raman spectrum of hemozoin with lexc. ¼ 647 nm.
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343 cm1 in the Raman spectra with lexc. ¼ 647 nm and
a different enhancement is no longer present in the spectra with
lexc. ¼ 676 nm (Fig. 4C1 and 4C2). Having the general advan-
tages of Raman spectroscopy in mind (no need for sample
preparation, applicable in a biological environment), these
results open up the possibility for the use of resonance Raman
spectroscopy as an analytical tool for studying the b-hematin
morphology.
The selective enhancement of the morphology-sensitive low
wavenumber Raman bands between 200 cm1 and 500 cm1 with
excitation wavelengths around lexc. ¼ 647 nm (Fig. 2) is caused
by a strong absorption band at 655 nm (Fig. 3). Again this band
is more intense in the spectrum of the more crystalline hemozoin
(Fig. 3A) compared with the spectrum of b-hematin (Fig. 3B).
The relative resonance enhancements in Fig. 2 are therefore
nicely explained by the absorption bands of hemozoin in Fig. 3,
which are presented, to the best of our knowledge, for the first
time.
In the following discussion particular attention should be
given to the enhancement of Raman modes in the low wave-
number region between 200 and 500 cm1. When comparing all
resonance excitation wavelengths (Fig. 2), the low wavenumber
modes of hemozoin show an increasing intensity from excitation
with lexc. ¼ 568 nm towards lexc. ¼ 633 nm, a maximal
enhancement with lexc. ¼ 647 nm, and again a decreasing
intensity with lexc. ¼ 676 nm. This enhancement pattern is in nice
accordance with the absorption spectrum (Fig. 3A).
The relative resonance enhancements of the Raman spectra of
hemozoin with lexc. ¼ 633 nm, 647 nm, and 676 nm are illustrated
in Fig. 4A1, 4B1, and 4C1 respectively and are compared with
the enhancement of the Raman modes of other hematin samples
in Fig. 4A2–4, 4B2–4, and 4C2–4. The wavenumber values of
the prominent Raman bands 405 cm1, 375 cm1, 368 cm1,
Analyst, 2009, 134, 1126–1132 | 1129

343 cm1, and 313 cm1 are given in Fig. 4B1. The modes at 368
cm1 and 343 cm1 are equally strongly enhanced with lexc. ¼ 633
nm (Fig. 4A1). The Raman band of hemozoin at 343 cm1 is
selectively enhanced with lexc. ¼ 647 nm compared with the
mode at 368 cm1 (Fig. 4B1). This relative resonance enhance-
ment is even more pronounced in the Raman spectrum with lexc.
¼ 676 nm, where the mode at 368 cm1 shows much less intensity
compared with the band at 343 cm1. The overall enhancement of
the low wavenumber modes in the Raman spectrum with lexc. ¼
676 nm is much smaller compared with lexc. ¼ 633 nm and lexc. ¼
647 nm. The normalized relative enhancement of the band at
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343 cm1 (E343) (relative to the band at 1623 cm1) in the reso-
nance Raman spectra of hemozoin (Hz) vs. b-hematin (bH), in
comparison with lexc. ¼ 647 nm and lexc. ¼ 676 nm, is about
a factor of two:
! where the bonded propionic acid side chains are connected (these
Hzð647Þ=bHð647Þ
E343 z2
Hzð676Þ=bHð676Þ
The relative enhancement of this Raman mode of Hz is caused
by the more crystalline morphology of the Hz sample compared
to the b-hematin sample. This relative resonance Raman
enhancement is reflected in a stronger absorption band at 655 nm
(Fig. 3).
The normal modes that belong to the most sensitive Raman
bands were assigned with help of density functional theory
(DFT) calculations of the Raman spectrum of the dimeric unit
cell of the hemozoin crystal (Fig. 1). The calculated atomic
displacements of the two Raman modes at 343 cm1 and 368
cm1 are illustrated in Fig. 5A and 5B, respectively.
The normal mode at 343 cm1 is a highly symmetric vibration
across the whole dimer and consists of two mirror-like parts in
both hematin units (Fig. 5A). A strong doming mode of the
tetrapyrrole backbone contributes to this vibration alongside an
Fig. 5 Atomic displacements of calculated vibrational modes of the dimeric unit cell of hemozoin. These two modes are assigned to the Raman bands
343 cm1 (A), and 368 cm1 (B), which are strongly enhanced with lexc. ¼ 647 nm (Fig. 2 and 3). These modes are marker bands for the sample
morphology due to their distinct relative resonance enhancement (Fig. 3), as discussed in the context.
1130 | Analyst, 2009, 134, 1126–1132
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out-of-phase movement of the central iron into the porphyrin
plane. Connected with this movement of the iron atom is
a stretching vibration of the Fe1–O41 iron–carboxylate linkage.
Further contributions are bending vibrations around the
porphyrin (doopCH, tCH2 vinyl, and rCH3 methyl) as well as
stretching and bending motions at the propionic acid side chains
(nCC, dCC, dCO40, tCH2). This Raman mode of hemozoin is
determined by the three-dimensional structure and therefore
likely to be sensitive for the sample morphology.
The normal mode at 368 cm1 is again a highly symmetric
vibration across the hemozoin dimer and symmetric in both
hematin units (Fig. 5B). The overall symmetry is disturbed due to
the iron–carboxylate bonds and the vibration is localized at the
propionate linkage. Obvious is furthermore a strong out-of-
plane wagging of the two pyrroles at the part of the porphyrins,
two pyrroles are marked with P1 in Fig. 5B). Further out-of-
plane bending motions of the methine bridges of the tetrapyrrole
and tCH2 contribute to this vibration as well as bending motions
in the propionate linkage (dCO41Fe1, dCO40) and around
the porphyrin (rCH3 methyl, tCH2 vinyl, and dCH methine).
The Raman mode at 368 cm1 is also very much determined by
the three-dimensional structure of hemozoin due to the locali-
zation around the propionate linkage and the strong out-of-
plane wagging of the two pyrroles P1. However, this vibration is
completely different compared with the mode at 343 cm1 and
the relative resonance Raman enhancements can therefore be
understood.
To verify these morphology-sensitive resonance Raman
enhancements of the low wavenumber modes, the Raman spectra
of hemozoin/b-hematin in Fig. 4A1, 4B1 and 4C1/4A2, 4B2 and
4C2 were compared to the Raman spectra of hematin (Fe(III)-
ProtoporphyrinIX-OH) as well as hemin in Fig. 4A3, 4B3 and
4C3 as well as Fig. 4A4, 4B4 and 4C4, respectively. The hematin
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sample shows a completely amorphous structure while hemin is
known to build large crystals (Teichmann crystals). Two samples
at the extreme ends of the morphology scale were therefore
available for the comparison with hemozoin/b-hematin. The low
wavenumber bands in the Raman spectra of hematin (Fig. 4A3,
4B3 and 4C3) are very weak compared with the spectra of
hemozoin (Fig. 4A1, 4B1 and 4C1). Furthermore, no significant
enhancement is observed for the resonance Raman spectrum of
hematin with lexc. ¼ 647 nm compared with the spectra with lexc.
¼ 633 nm and lexc. ¼ 676 nm. In contrast, a strong enhancement
of the low wavenumber modes in the resonance Raman spectra
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of hemin is seen in Fig. 4A4, 4B4. The strongest enhancement of
these morphology-sensitive Raman modes of hemin is present
in the spectrum with lexc. ¼ 633 nm (Fig. 4A4). A strong
enhancement is also observed for excitation with lexc. ¼ 647 nm
(Fig. 4B4), while no enhancement is seen at all for lexc. ¼ 676 nm
(Fig. 4C4). These comparative results of the resonance Raman
spectra of hemozoin and b-hematin with the spectra of hematin
and hemin very nicely prove the usefulness of resonance Raman
micro-spectroscopy with lexc. ¼ 647 nm as a probe of the
morphology of hematin samples.
Conclusion and outlook
This study demonstrates the potential of resonance Raman
spectroscopy as an analytical tool for investigating the
morphology of hematin samples. Hemozoin was extracted from
Plasmodium falciparum-infected erythrocytes and b-hematin was
synthesized by the dehydration of hematin (Fe(III)Protopor-
phyrinIX-OH).35,36
Distinct relative resonance Raman enhancements of low
wavenumber modes of hemozoin were elucidated with the help of
excitation wavelengths at 568 nm, 633 nm, 647 nm, and 676 nm
(Fig. 2 and 4). It was shown that the Raman modes at 343 cm1
and 368 cm1 are selectively enhanced (Fig. 4). These normal
modes were assigned using DFT calculations of the hemozoin
dimer (Fig. 5). The mode at 343 cm1 in the Raman spectrum of
hemozoin, which is strongly enhanced with lexc. ¼ 647 nm
(Fig. 4B1), was assigned to combined, highly symmetric doming
modes of the two hematin units in the hemozoin dimer (Fig. 5A).
The Raman band at 368 cm1 was assigned to a normal mode
localized around the propionate linkage (Fig. 5B) with strong
out-of-plane wagging contributions of the two pyrroles P1
(where the propionic acid side chains of the reciprocal propio-
nate linkages of the two hematins in the hemozoin dimer are
connected). These bands are stronger in the resonance Raman
spectrum of extracted hemozoin (lexc. ¼ 647 nm) (Fig. 4B1)
compared to a less crystalline b-hematin (Fig. 4B2). The
comparison to a very crystalline sample of hemin (Teichmann
crystals) and an amorphous sample of hematin has proven the
selective enhancement of the morphology-sensitive Raman
modes with lexc. ¼ 633 nm and lexc. ¼ 647 nm (Fig. 4).
The UV-VIS-NIR absorption spectrum of hemozoin explains
this selective resonance enhancement due to the appearance of
a strong absorption band at 655 nm (Fig. 3). Also the dominant
resonance enhancement of the mode at 1372 cm1 in the Raman
spectrum of hemozoin with lexc. ¼ 830 nm (Fig. 2) was under-
stood on the basis of a broad absorption band around 870 nm
(Fig. 3).
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The demonstrated capabilities of resonance Raman spectros-
copy lead to a better understanding of the hemozoin structure
and will contribute to the elucidation of the interference of
antimalarial drugs with the crystallization process of the malaria
pigment.
Acknowledgements
The authors gratefully acknowledge that computations were run
at the computing centre of University Leipzig and for the tech-
nical assistance of Elisabeth Fischer in cell culture. T. F. grate-
fully acknowledges financial support by the German Chemical
Society (section analytical chemistry) for a lecture trip to the
SPEC conference. Financial support by the German Research
Foundation (SFB 535, TP A12) to K. B. is highly acknowledged.
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