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Morphology-sensitive Raman modes of the malaria pigment hemozoin


Torsten Frosch,*ab Sasa Koncarevic,c Katja Beckerc and Jrgen Poppab u
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 633 nm and l 647 nm. 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. 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. 830 nm. In conclusion, resonance Raman micro-spectroscopy with lexc. 647 nm was shown to have great potential as an analytical tool to probe the morphology of hematin samples.

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Introduction
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.13 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 subsequently detoxied 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 detoxication process,611 most probably by interaction with hemozoin.12,13 Chloroquine and other quinoline antimalarials 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 antimalarial drugs with the important intracellular target hemozoin.
a Institut fr Physikalische Chemie, Friedrich-Schiller-Universitt Jena, u a Helmholtzweg 4, D-07743 Jena, Germany. E-mail: torsten.frosch@gmx. de; Tel: +49 3641 948351 b Institut fr Photonische Technologien e.V., Albert-Einstein-Strae 9, u D-07745 Jena, Germany c Interdisziplinres Forschungszentrum, Universitt Giessen, Heinrich-Buffa a 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 Sao Jos dos Campos, Sao Paulo, Brazil, October 2529, 2008. e

To study this aspect, Raman spectroscopy1416 has unique potential. It can be employed for a fast, label-free and noninvasive investigation of drugtarget-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 conventional light microscope allows for a resonance Raman spectroscopic 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 meoquine24 and quinine25 can be monitored in therapeutically meaningful concentrations with the help of the UV resonance enhancement of the Raman spectra. Also the inuence 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 ndings.2228 Furthermore, the malaria pigment hemozoin could be localized in infected red blood cells using resonance Raman micro-spectroscopy.22,2931 Single erythrocytes were scanned with a spatial resolution of 0.5 mm, and Raman images of the hemozoin distribution in P. falciparum were built on the basis of the spectral array.22 The binding afnity 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 wavenumber 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 antimalarial 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 structural study on hemozoin. Resonance Raman micro-spectroscopy was applied to selectively enhance morphology-sensitive Raman modes of different hematin samples. The results further support the great potential of resonance Raman micro-spectroscopy as an analytical tool in malaria research.

with water until the ltrate 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

Materials and methods


Extraction of hemozoin Blood stages of the P. falciparum strain Dd2 (chloroquineresistant) were maintained in culture using a modied 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 chloroformmethanol (2 : 1 v/v) and chloroformmethanolwater (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 proteinlinked 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 reux. Propionic acid was added until the pH reached 4.1. During the addition of the rst 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 reux. After cooling at ambient temperature the reaction mixture was ltered through a nylon lter (Millipore, 22 mm), the solid was washed alternating with methanol and water and nally with 0.1 M NaHCO3 solution. The procedure was nished by washing
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Results and discussion


As explained in the Introduction the malaria pigment hemozoin is a major target for antimalarial drugs.1013,22,4045 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 Fe1O41 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 drugtarget-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 hematin(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)ProtoporphyrinIX-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 Fe1O41 ironcarboxylate 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 investigated 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. 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. falciparum.22,34 The Raman spectra with excitation wavelengths lexc. 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. 647 nm. The comparison of the different resonance Raman spectra shows some dramatic selective mode enhancements most prominently the enhancement 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 inuenced by the closely packed porphyrins in the three-dimensional structure of hemozoin. This effect is an aggregation enhancement.44,50 These resonance effects in the Raman spectra of the aggregated hemozoin originate from electronic transitions. Signicant 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. 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. 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. 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 propionate 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 bhematin sample (Fig. 3B). Some recent results derived with non-resonant Raman spectroscopy22 lead the interest to a comparison of the resonance Raman spectra of hemozoin (Fig. 4A1, 4B1, and 4C1) with bhematin 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 signicant 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 signicantly stronger resonance enhancement of these bands in the Raman spectra of hemozoin (Fig. 4A1 and 4B1) with excitation 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

343 cm1 in the Raman spectra with lexc. 647 nm and a different enhancement is no longer present in the spectra with lexc. 676 nm (Fig. 4C1 and 4C2). Having the general advantages 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. 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 rst time. In the following discussion particular attention should be given to the enhancement of Raman modes in the low wavenumber 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. 568 nm towards lexc. 633 nm, a maximal enhancement with lexc. 647 nm, and again a decreasing intensity with lexc. 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. 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. 4A24, 4B24, and 4C24. The wavenumber values of the prominent Raman bands 405 cm1, 375 cm1, 368 cm1,

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. 633 nm, B: lexc. 647 nm, and C: lexc. 676 nm. Some prominent wavenumber values are given in the resonance Raman spectrum of hemozoin with lexc. 647 nm.

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343 cm1, and 313 cm1 are given in Fig. 4B1. The modes at 368 cm1 and 343 cm1 are equally strongly enhanced with lexc. 633 nm (Fig. 4A1). The Raman band of hemozoin at 343 cm1 is selectively enhanced with lexc. 647 nm compared with the mode at 368 cm1 (Fig. 4B1). This relative resonance enhancement is even more pronounced in the Raman spectrum with lexc. 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. 676 nm is much smaller compared with lexc. 633 nm and lexc. 647 nm. The normalized relative enhancement of the band at 343 cm1 (E343) (relative to the band at 1623 cm1) in the resonance Raman spectra of hemozoin (Hz) vs. b-hematin (bH), in comparison with lexc. 647 nm and lexc. 676 nm, is about a factor of two: ! Hz647=bH647 z2 E343 Hz676=bH676 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 reected 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

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 Fe1O41 ironcarboxylate 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 ironcarboxylate bonds and the vibration is localized at the propionate linkage. Obvious is furthermore a strong out-ofplane wagging of the two pyrroles at the part of the porphyrins, where the bonded propionic acid side chains are connected (these two pyrroles are marked with P1 in Fig. 5B). Further out-ofplane 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 localization around the propionate linkage and the strong out-ofplane 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

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. 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.

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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 signicant enhancement is observed for the resonance Raman spectrum of hematin with lexc. 647 nm compared with the spectra with lexc. 633 nm and lexc. 676 nm. In contrast, a strong enhancement of the low wavenumber modes in the resonance Raman spectra 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. 633 nm (Fig. 4A4). A strong enhancement is also observed for excitation with lexc. 647 nm (Fig. 4B4), while no enhancement is seen at all for lexc. 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. 647 nm as a probe of the morphology of hematin samples.

The demonstrated capabilities of resonance Raman spectroscopy 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 technical assistance of Elisabeth Fischer in cell culture. T. F. gratefully acknowledges nancial 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.

References
1 http://rbm.who.int/wmr2005. 2 R. W. Snow, C. A. Guerra, A. M. Noor, H. Y. Myint and S. I. Hay, Nature, 2005, 434, 214. 3 J. Sachs and P. Malaney, Nature, 2002, 415, 680. 4 I. M. Hastings, P. G. Bray and S. A. Ward, Science, 2002, 298, 74. 5 T. E. Wellems, Science, 2002, 298, 124. 6 R. G. Ridley, Nature, 2002, 415, 686. 7 M. Foley and L. Tilley, Pharmacol. Ther., 1998, 79, 55. 8 S. E. Francis, D. J. Sullivan Jr. and D. E. Goldberg, Annu. Rev. Microbiol., 1997, 51, 97. 9 L. M. B. Ursos and P. D. Roepe, Medicinal Research Reviews, 2002, 22, 465. 10 D. J. Sullivan, Jr., H. Matile, R. G. Ridley and D. E. Goldberg, J. Biol. Chem., 1998, 273, 31103. 11 D. J. Sullivan, Jr., I. Y. Gluzman, D. G. Russell and D. E. Goldberg, Proc. Natl. Acad. Sci. USA, 1996, 93, 11865. 12 S. Pagola, P. W. Stephens, D. S. Bohle, A. D. Kosar and S. K. Madsen, Nature, 2000, 404, 307. 13 R. Buller, M. L. Peterson, O. Almarsson and L. Leisirowitz, Crystal Growth & Design, 2002, 2, 553. 14 D. A. Long, The Raman Effect, Wiley & Sons, New York, 2002. 15 Handbook of Vibrational Spectroscopy, ed. J. M. Chalmers and P. R. Grifths, Wiley & Sons, Chichester, 2002. 16 M. Schmitt and J. Popp, J. Raman Spectrosc., 2006, 37, 20. 17 T. Frosch, B. K stner, S. Schl cker, A. Szeghalmi, M. Schmitt, u u W. Kiefer and J. Popp, J. Raman Spectrosc., 2004, 35, 819. 18 T. Frosch, T. Meyer, M. Schmitt and J. Popp, Anal. Chem., 2007, 79, 6159. 19 Biological Applications of Raman Spectroscopy, ed. T. G. Spiro, Wiley & Sons, New York, 1988, vol. 13, and refs cited therein. 20 R. Petry, M. Schmitt and J. Popp, ChemPhysChem, 2003, 4, 14. 21 G. J. Puppels, F. F. M. De Mul, C. Otto, J. Greve, M. RobertNicoud, D. J. Arndt-Jovin and T. M. Jovin, Nature, 1990, 347, 301. 22 T. Frosch, S. Koncarevic, L. Zedler, M. Schmitt, K. Schenzel, K. Becker and J. Popp, J. Phys. Chem. B, 2007, 111, 11047. 23 T. Frosch, M. Schmitt, G. Bringmann, W. Kiefer and J. Popp, J. Phys. Chem. B, 2007, 111, 1815. 24 T. Frosch, M. Schmitt and J. Popp, Anal. Bioanal. Chem., 2007, 387, 1749. 25 T. Frosch, M. Schmitt and J. Popp, J. Phys. Chem. B, 2007, 111, 4171. 26 T. Frosch, M. Schmitt, T. Noll, G. Bringmann, K. Schenzel and J. Popp, Anal. Chem., 2007, 79, 986. 27 T. Frosch, M. Schmitt, K. Schenzel, J. H. Faber, G. Bringmann, W. Kiefer and J. Popp, Biopolymers, 2006, 82, 295. 28 T. Frosch and J. Popp, Journal of Molecular Structure, 2009, 924926, 301308. 29 C. W. Ong, Z. X. Shen, K. H. H. Ang, U. A. K. Kara and S. H. Tang, Appl. Spectrosc., 1999, 53, 1097. 30 C. W. Ong, Z. X. Shen, K. H. H. Ang, U. A. K. Kara and S. H. Tang, Appl. Spectrosc., 2002, 56, 1126. 31 B. R. Wood, S. J. Langford, B. M. Cooke, F. K. Glenister, J. Lim and D. McNaughton, FEBS Letters, 2003, 554, 247. 32 W. Trager and J. B. Jensen, Science, 1976, 193, 673.

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)ProtoporphyrinIX-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. 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 propionate linkages of the two hematins in the hemozoin dimer are connected). These bands are stronger in the resonance Raman spectrum of extracted hemozoin (lexc. 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. 633 nm and lexc. 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. 830 nm (Fig. 2) was understood on the basis of a broad absorption band around 870 nm (Fig. 3).
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33 S. L. Cranmer, C. Magowan, J. Liang, R. L. Coppel and B. M. Cooke, Trans. R. Soc. Trop. Med. Hyg., 1997, 91, 363. 34 M. Jaramillo, D. C. Gowda, D. Radzioch and M. J. Olivier, Immunology, 2003, 171, 4243. 35 D. S. Bohle, B. J. Conklin, D. Cox, S. K. Madson, S. Paulson, P. W. Stephens and G. T. Yee, ACS Symposium Ser., 1994, 572, 497, and refs cited therein. 36 T. J. Egan, W. W. Mavuso and K. K. Ncokazi, Biochemistry, 2001, 40, 204. 37 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, J. A. Montgomery, Jr, T. Vreven, K. N. Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G. A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox, H. P. Hratchian, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth, P. Salvador, J. J. Dannenberg, V. G. Zakrzewski, S. Dapprich, A. D. Daniels, M. C. Strain, O. Farkas, D. K. Malick, A. D. Rabuck, K. Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul, S. Clifford, J. Cioslowski, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara, M. Challacombe, P. M. W. Gill, B. Johnson, W. Chen, M. W. Wong, C. Gonzalez and J. A. Pople, Gaussian 03, Revision C.02, Gaussian, Inc., Wallingford, CT, 2004. A. D. Becke, Phys. Rev. A, 1988, 38, 3098. J. P. Perdew and W. Yue, Phys. Rev. B, 1986, 33, 8800. T. J. Egan, Targets, 2003, 2, 115. M. O. Senge and S. Hatschler, ChemBioChem, 2000, 1, 247. A. Dorn, H. Stoffel, H. Mathile, R. Bubendorf and R. G. Ridley, Nature, 1995, 374, 269. I. Solomonov, M. Osipova, Y. Feldman, C. Baehtz, K. Kjaer, I. K. Robinson, G. T. Webster, D. McNaughton, B. Wood, I. Weissbuch and J. Leiserowitz, J. Am. Chem. Soc., 2007, 129, 2615. B. R. Wood, S. J. Langford, B. M. Cooke, J. Lim, F. K. Glenister, M. Duriska, J. K. Unthank and D. McNaughton, J. Am. Chem. Soc., 2004, 126, 9233. B. R. Wood and D. McNaughton, Expert Rev. Proteomics, 2006, 3, 525. A. F. G. Slater, W. J. Swiggard, B. R. Orton, W. D. Flitter, D. E. Goldberg, A. Cerami and G. B. Henderson, Proc. Natl. Acad. Sci. USA, 1991, 88, 325. D. S. Bohle, R. E. Dinnebier, S. K. Madsen and P. W. Stephens, J. Biochem., 1997, 272, 713. D. S. Bohle and J. Helms, Biochem. Biophys. Res. Commun., 1993, 193, 504. D. S. Bohle, A. D. Kosar and P. W. Stephens, Acta Cryst. D, 2002, 58, 1752. D. L. Akins, S. Ozcelic, H. R. Zhu and C. Guo, J. Phys. Chem., 1997, 101, 3251.

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