Browsing by Author "Ncokazi, Kanyile K"
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- ItemRestrictedCrystallization of synthetic haemozoin (beta-haematin) nucleated at the surface of lipid particles(Royal Society of Chemistry, 2010) Hoang, Anh N; Ncokazi, Kanyile K; de Villiers, Katherine A; Wright, David W; Egan, Timothy JThe mechanism of formation of haemozoin, a detoxification by-product of several blood-feeding organisms including malaria parasites, has been a subject of debate; however, recent studies suggest that neutral lipids may serve as a catalyst. In this study, a model system consisting of an emulsion of neutral lipid particles was employed to investigate the formation of b-haematin, the synthetic counterpart of haemozoin, at the lipid–water interface. A solution of monoglyceride, either monostearoylglycerol (MSG) or monopalmitoylglycerol (MPG), dissolved in acetone and methanol was introduced to an aqueous surface. Fluorescence, confocal and transmission electron microscopic (TEM) imaging and dynamic light scattering analysis of samples obtained from beneath the surface confirmed the presence of homogeneous lipid particles existing in two major populations: one in the low micrometre size range and the other in the hundred nanometre range. The introduction of haem (Fe(III)PPIX) to this lipid particle system under biomimetic conditions (37 ◦C, pH 4.8) produced b-haematin with apparent first-order kinetics and an average half life of 0.5 min. TEM of monoglycerides (MSG or MPG) extruded through a 200 nm filter with haem produced b-haematin crystals aligned and parallel to the lipid–water interface. These TEM data, together with a model system replacing the lipid with an aqueous organic solvent interface using either methyl laurate or docosane demonstrated that the OH and C=O groups are apparently necessary for efficient nucleation. This suggests that b-haematin crystallizes via epitaxial nucleation at the lipid–water interface through interaction of Fe(III)PPIX with the polar head group. Once nucleated, the crystal grows parallel to the interface until growth is terminated by the curvature of the lipid particle. The hydrophobic nature of the mature crystal favours an interior transport resulting in crystals aligned parallel to the lipid–water interface and each other, strikingly similar to that seen in malaria parasites.
- ItemOpen AccessInteractions of quinoline antimalarials with haematin and their effect on ß-haematin formation(2005) Ncokazi, Kanyile K; Egan, Timothy JThermodynamic compensation in the interaction of quinoline antimalarials with haematin in 40% (v/v) aqueous OMSO has been compared with that in pure aqueous solution. The data indicate that the degree of desolvation and loss of conformational freedom is identical in both systems. The nature of interactions between quinoline drugs and haematin was investigated spectroscopically in organic and mixed solvents. Free energies of association of chloroquine, quinine and quinidine with haematin are largely insensitive to the increasing concentration of NaCI0. This demonstrates that electrostatic interactions play a minor role in the overall stability of these complexes under these conditions. Increasing concentration of OM SO weakens association of chloroquine, amodiaquine, quinine, quinidine and 9-epiquinine with haematin. These effects suggest that the interactions are hydrophobic. Furthermore, it has been demonstrated that free energy of association with haematin weakens as a function of decreasing solvent polarity in organic solvents. However, free energies of association are weaker in mixed aqueous solvents than in pure organic solvents. This indicates that dispersion and electrostatic interactions are relatively stronger in a non-aqueous environment. A new assay has been developed for measuring inhibition of ,ß-haematin formation using 5% (v/v) aqueous pyridine solution. This pyridine solution forms a low complex with haematin in aqueous solution but not with ,ß-haematin. Formation of ß-haematin is brought about in 4.5 M acetate, pH 4.5 at 60°C. The assay is rapid (60 min incubation) and requires no centrifugation or expensive radioactive material. This assay is compatible with high throughput screening and analysis can be done by visual inspection of ,ß- haematin inhibitors. The IC⁵⁰ values obtained were compared with those reported in other assays for 13 compounds investigated. There was generally a good correlation between the pyridine assay and other assays. Used qualitatively, the method was also employed for screening 48 compounds for ,ß-haematin inhibition. All of these compounds produced results in agreement with expectation from previous investigations.
- ItemRestrictedThe mechanism of beta-hematin formation in acetate solution. Parallels between hemozoin formation and biomineralization processes(American Chemical Society, 2001) Egan, Timothy J; Mavuso, Winile W; Ncokazi, Kanyile KFormation of â-hematin in acidic acetate solution has been investigated using quantitative infrared spectroscopy, X-ray diffraction, and scanning and transmission electron microscopy. The process occurs via rapid precipitation of amorphous (or possibly nanocrystalline) hematin, followed by slow conversion to crystalline â-hematin. Definitive evidence that the reaction occurs during incubation in acetate medium, rather than during the drying stage, is provided by X-ray diffraction and infrared spectroscopy of the wet material. The reaction follows a sigmoidal function indicative of a process of nucleation and growth and was modeled using the Avrami equation. Reaction rates and the dimensionality of growth (as indicated by the value of the Avrami constant) are strongly influenced by stirring rate. The reaction follows Arrhenius behavior, and there is a strong dependence of both the rate constant and the Avrami constant on acetate concentration. Acetate may act as a phase transfer catalyst, solubilizing hematin and facilitating its redeposition as â-hematin. The pH dependence of the process indicates that only the monoprotonated species of hematin is active in forming â-hematin. The formation of â-hematin closely parallels many mineralization processes, and this suggests that hemozoin formation may be a unique biomineralization process. Inferences are drawn with respect to the formation of hemozoin in vivo.
- ItemRestrictedQuinoline antimalarials decrease the rate of beta-hematin formation(Elsevier, 2005) Egan, Timothy J; Ncokazi, Kanyile KThe strength of inhibition of b-hematin (synthetic hemozoin or malaria pigment) formation by the quinoline antimalarial drugs chloroquine, amodiaquine, quinidine and quinine has been investigated as a function of incubation time. In the assay used, b-hematin formation was brought about using 4.5 M acetate, pH 4.5 at 60 C. Unreacted hematin was detected by formation of a spectroscopically distinct low spin pyridine complex. Although, these drugs inhibit b-hematin formation when relatively short incubation times are used, it was found that b-hematin eventually forms with longer incubation periods (8 h for quinine). This conclusion was supported by both infrared and X-ray powder diffraction observations. It was further found that the IC50 for inhibition of b-hematin formation increases markedly with increasing incubation times in the case of the 4-aminoquinolines chloroquine and amodiaquine. By contrast, in the presence of the quinoline methanols quinine and quinidine the IC50 values increase much more slowly. This results in a partial reversal of the order of inhibition strengths at longer incubation times. Scanning electron microscopy indicates that b-hematin crystals formed in the presence of chloroquine are more uniform in both size and shape than those formed in the absence of the drug, with the external morphology of these crystallites being markedly altered. The findings suggest that these drugs act by decreasing the rate of hemozoin formation, rather than irreversibly blocking its formation. This model can also explain the observation of a sigmoidal dependence of b-hematin inhibition on drug concentration.