In situ localization and structural analysis of the malaria pigment hemozoin

Raman microspectroscopy was applied for an in situ localization of the malaria pigment hemozoin in Plasmodium falciparum-infected erythrocytes. The Raman spectra (lambda(exc) = 633 nm) of hemozoin show very intense signals with a very good signal-to-noise ratio. These in situ Raman signals of hemozoin were compared to Raman spectra of extracted hemozoin, of the synthetic analogue beta-hematin, and of hematin and hemin. beta-Hematin was synthesized according to the acid-catalyzed dehydration of hematin and the anhydrous dehydrohalogenation of hemin which lead to good crystals with lengths of about 5-30 mu m. The Raman spectra (lambda(exc) = 1064 nm) of hemozoin and beta-hematin show almost identical behaviors, while some low wavenumber modes might be used to distinguish between the morphology of differently synthesized beta-hematin samples. The intensity pattern of the resonance Raman spectra (lambda(exc), = 568 nm) of hemozoin and beta-hematin differ significantly from those of hematin and hemin. The most striking difference is an additional band at 1655 cm(-1) which was only observed in the spectra of hemozoin and beta-hematin and cannot be seen in the spectra of hematin and hemin. Raman spectra of the beta-hematin dimer were calculated ab initio (DFT) for the first time and used for an assignment of the experimentally derived Raman bands. The calculated atomic displacements provide valuable insight into the most important molecular vibrations of the hemozoin dimer. With help from these DFT calculations, it was possible to assign the Raman band at 1655 cm(-1) to a mode located at the propionic acid side chain, which links the hemozoin dimers to each other. The Raman band at 1568 cm(-1), which has been shown to be influenced by an attachment of the antimalarial drug chloroquine in an earlier study, could be assigned to a C=C stretching mode spread across one of the porphyrin rings and is therefore expected to be influenced by a pi-pi-stacking to the drug.