The role of lipid in malaria pigment (Haemozoin) formation under biomimetic conditions

 

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dc.contributor.advisor Egan, Timothy J en_ZA
dc.contributor.author Ambele, Melvin Anyasi en_ZA
dc.date.accessioned 2014-10-21T14:23:22Z
dc.date.available 2014-10-21T14:23:22Z
dc.date.issued 2013 en_ZA
dc.identifier.citation Ambele, M. 2013. The role of lipid in malaria pigment (Haemozoin) formation under biomimetic conditions. University of Cape Town. en_ZA
dc.identifier.uri http://hdl.handle.net/11427/8721
dc.description Includes bibliographical references. en_ZA
dc.description.abstract Previous studies have proposed lipids and/or proteins to be involved in the biomineralization process of haemozoin formation in the malaria parasite which is a target for many antimalarial drugs. This study therefore investigated the biomolecules involved and established the role of lipids in mediating Hz formation under biomimetic conditions. Hz crystals were isolated from mature trophozoites obtained from saponin-lysed iRBCs. Trophozoites were triturated to release digestive vacuoles and subjected to freeze/thaw cycles to release Hz. Supernatant from SDS-washed Hz crystals and solution of dissolved crystals in NaOH were used for SDS PAGE and LC-MS/MS protein identification. None of the proteins HRPII, HDP, or those belonging to the proposed protein complex previously suggested to be involved in Hz formation in vivo, were identified in the proteome associated with Hz crystals. In addition, no 'stand out' proteins with expected Hz-mediating activity in the malaria parasite were identified. Contrastingly, putative lipoproteins and uncharacterized proteins with roles in lipid binding and transport were identified for the first time in the proteome associated with Hz crystals. Lipids consisting of MSG, MPG, DPG, DLG and DOG (NLB) previously reported to be associated with Hz crystals were easily identified to be associated with Hz crystals. Lipid was also found to be in vastly greater abundance than proteins in the Hz crystal. The absence, therefore, of all proteins previously suggested to be involved in Hz formation from the proteome associated with the Hz crystal, coupled with the identification of lipoproteins and the easily detected abundance of lipids with Hz-mediating activity, strongly suggest that lipids are the biomolecules responsible for the biomineralization of Hz formation in vivo. A solution mixture of NLB and haematin both prepared in acetone/methanol deposited on citrate buffer (50 mM, pH 4.8, 37oC), was allowed to incubate at 37oC for different lengths of time to investigate β-haematin formation. The product formed was confirmed by FT-IR to be β-haematin. The reaction was very fast giving yields of about 90% at 2.15 and 1.07 lipid-tohaem mol ratios and was completed in less than 10 min. The reaction only slowed when there were two haem molecules to one fatty acid chain, but still gave yields of about 90% in under 30 min. On the other hand, this slower kinetics allowed for the effects of relevant biological ions on the process to be investigated. Relevant biological ions at concentrations which they exist in RBC and/or blood are unlikely to greatly affect the lipid mediated process of Hz formation in vivo as only a 2.3 fold decrease in kinetics was observed in the presence of these ions. β-Haematin formed over time during the reaction showed an increase in fluorescence measured using confocal laser microscopy. This qualitatively corresponds to that of bulk kinetic measurements and also demonstrates the formation of well-ordered crystals in situ in association with the lipid component of the emulsion. However, the β-haematin crystals formed seemed to have a distinct external morphology different from Hz itself and thus this was investigated further. Solutions of NLB and haematin in acetone/methanol were mixed by means of vortexing, ultrasonication or micromixing before being used for β-haematin formation reactions. The β-haematin crystals formed at the emulsion layer as well as lipid droplets were characterized by TEM, TEM-ED and ESI/EELS and were compared to Hz. β-Haematin crystals formed from the vortexed mixture appeared to be smaller in size with external morphology and hkl indices different from Hz while ultrasonication or micromixing produced very long β-haematin crystals that are identical to Hz both in overall crystal habit and form. An observed difference in texture between the β-haematin crystals and Hz was likely due to the presence of proteins and lipids on the Hz crystal as shown in the proteomic and lipid studies of this work. The external morphology of the β-haematin formed was not affected by the NLB-to-haematin ratio at any given time nor by the kinetics of the crystal growth process. β-Haematin crystals formed at the emulsion layer were always seen to make contact with non-hollow lipid droplets with continuous hydrophobic interiors via their {100} faces. The lipid droplets in contact with crystals were found to control the length of the crystal. Growth stopped when the angle of contact between the lipid and crystal reached approximately 38o. The invariable observation of β-haematin crystals with their {100} face in contact with lipid droplets that control their sizes strongly suggests that lipids act as the nucleator, providing a favourable microenvironment suitable for the interaction with the propionic acid groups at the {100} surface of the crystal via the glycerol –OH group of the lipid. The proposal that lipids are the sole biomolecules responsible for Hz formation in vivo is strongly supported by the following findings: Firstly, all the proteins previously suggested to be involved in Hz formation were absent from the proteome associated with Hz. Interestingly, it was lipids that were found to be in relative abundance over these proteins in the Hz crystal. Secondly, these lipids were highly efficient in mediating β-haematin formation under biomimetic conditions producing crystals that were identical in overall habit and form to that of Hz itself. Finally, the lipid droplets invariably observed in contact with the crystals, controls their size and probably nucleate crystal growth via their {100} faces. en_ZA
dc.language.iso eng en_ZA
dc.title The role of lipid in malaria pigment (Haemozoin) formation under biomimetic conditions en_ZA
dc.type Thesis / Dissertation en_ZA
uct.type.publication Research en_ZA
uct.type.resource Thesis en_ZA
dc.publisher.institution University of Cape Town
dc.publisher.faculty Faculty of Science en_ZA
dc.publisher.department Department of Chemistry en_ZA
dc.type.qualificationlevel Doctoral en_ZA
dc.type.qualificationname PhD en_ZA
uct.type.filetype Text
uct.type.filetype Image


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