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Hematin crystallization from aqueous and organic solvents
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10.1063/1.4816106
/content/aip/journal/jcp/139/12/10.1063/1.4816106
http://aip.metastore.ingenta.com/content/aip/journal/jcp/139/12/10.1063/1.4816106
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Figures

Image of FIG. 1.
FIG. 1.

Schematic illustration of infecting a red blood cell. Hemoglobin is brought into the parasite's digestive vacuole (DV), where it is catabolized, releasing four hemes per each processed hemoglobin molecule. The heme converts to toxic hematin, which is sequestered into hemozoin crystals. Spherical agglomerations of neutral lipids that are several hundred nanometers in size, referred to as nanospheres, are present in the digestive vacuole. Numbers mark possible locations for hemozoin crystal formation: 1—in the bulk of aqueous phase, 2—in the bulk of the lipid nanospheres, 3—at lipid/aqueous interface, 4—at the DV membrane in the lipid phase, and 5—at the DV membrane in the aqueous phase.

Image of FIG. 2.
FIG. 2.

Illustration of the experimental procedures. (a) Crystallization of hematin at the water-octanol interface. (b) Determination of the extinction coefficient of hematin in water-saturated octanol.

Image of FIG. 3.
FIG. 3.

Towards the determination of the concentration of hematin in water-saturated octanol. (a) In 20 vials, 2.00 ml of hematin/NaOH solution with pH = 12.78 is kept for two weeks in contact with 2.00 ml of octanol. The concentration of hematin in the octanol phase is determined from the difference of its concentration in the NaOH solution prior and post exchange with the octanol. (b) Determination of the extinction coefficient ɛ of hematin in a 0.10 M NaOH solution at 607 nm, indicated by an arrow in spectrum in the inset. Results of three independent determinations are shown. (c) Determination of the extinction coefficient ɛ of hematin in water-saturated octanol at 594 nm, indicated by an arrow in spectrum in the inset. Results of two independent determinations are shown. In (c) and (d), ɛ is the slope of the solid straight line through the data points.

Image of FIG. 4.
FIG. 4.

Aggregation of hematin in aqueous solutions. (a) and (b) The intensity correlation functions of light scattered from 55 M hematin solutions in phosphate buffer in (a) and acetate buffer in (b); the respective pH values are indicated in the plots. (c) and (d) The intensity distribution functions corresponding to the (a) and (b), respectively. The peaks with characteristic diffusion times of ∼12 ms correspond to the monomers or dimers; those with times ∼1 ms correspond to aggregates that are 80–100× larger. A signify the areas of the respective aggregate peaks.

Image of FIG. 5.
FIG. 5.

The solubility of hematin in phosphate solution as a function of pH. Concentrations greater than 0.5 M were determined spectrophotometrically; those lower—by an enzymatic assay, as discussed in the text.

Image of FIG. 6.
FIG. 6.

The surface structure of β-hematin crystals grown in aqueous solutions at pH = 4.8 in 0.1 M acetate buffer. (a)–(f) are phase AFM images collected in tapping mode. (a) Low magnification view of two twinned crystals; dashed lines highlight twin boundary. (b) High magnification view of the surface of a crystal showing rounded formations. (c) A crystal with apparent lamellar structure, indicated by arrows. (d) High resolution image of area highlighted by white square in (c) shows rounded formations similar to those in (b). (e) A crystal with a relatively smooth surface around the white square and a rough area covering more than 80% of the top face. (f) A high resolution image of area highlighted by a square in (e) showing that the apparently smooth area consists of lamellar layers. (g) Surface profile along dashed line from a height image of the crystal seen in (e). The thicknesses of the lamellar layers are shown in nanometers.

Image of FIG. 7.
FIG. 7.

The morphology of β-hematin crystals grown at the interface between aqueous citrate buffer with pH = 4.8 and water-saturated octanol. (a) and (b) are phase AFM images collected in contact mode. (a) Low magnification AFM view of a crystal revealing sharp edges and smooth faces. Miller indices are assigned to the crystal faces as discussed in the text. (b) High magnification image of the area highlighted with a square in (a). (c) Low magnification scanning electron microscopy (SEM) image of clusters seen in supersaturated hematin solutions. (d) High magnification SEM image of a cluster, seen at low magnification in the inset, demonstrating that the clusters consist of crystals of size several hundred nanometers and rougher formations, which are likely amorphous aggregates.

Image of FIG. 8.
FIG. 8.

The solubility of hematin in water-saturated octanol at three temperatures. (a) Vials of water-saturated octanol kept at 25 °C in contact with hematin amounts that would yield, upon full dissolution, total concentrations of 0, 0.1, 0.2, …, 1.0 mM, from left to right. The solid residue at the bottom of all vials with non-zero hematin amounts indicates that full dissolution was not achieved. (b)–(d) The evolution over four weeks of the concentration of hematin dissolved in octanol as a function of the amount of hematin added to each vial at 9 °C, 25 °C, and 45 °C as indicated in the plots; two determinations were carried out at 25 °C. The solution concentration, which does not increase upon increasing the total amount of hematin in the vials nor change over time, is taken as solubility and is indicated with a horizontal dashed line. (e) The temperature dependence of the solubility of hematin in water-saturated octanol from the data in (b)–(d).

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/content/aip/journal/jcp/139/12/10.1063/1.4816106
2013-07-25
2014-04-24
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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: Hematin crystallization from aqueous and organic solvents
http://aip.metastore.ingenta.com/content/aip/journal/jcp/139/12/10.1063/1.4816106
10.1063/1.4816106
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