PEGs are able to induce βH formation in acid conditions.

<p>Spontaneous heme crystallization was performed in the presence of 4.7% of different PEGs at 100 µM, in 0.5 M sodium acetate buffer pH 4.8, over 5 days at 28°C with a final volume of 1.0 mL. Samples were centrifuged and the pellet washed in 0.1 M sodium bicarbonate buffer and 2.5% SDS, pH 9.1, until the solution was almost clear. (A) Pellets were then characterized by FTIR spectroscopy. The large Nujol peaks in the region between 1300 cm⁻¹ and 1600 cm⁻¹ are obscured by the labels, but the key βH peaks are clearly seen at 1664 cm⁻¹ and 1210 cm⁻¹. (B) X-ray powder diffraction (XRD) confirms the identity of βH.</p

Values of r² for different values of n and rate constants for β-hematin formation in the presence of PEGs.

a<p>n = 4 for PEG 3.350 and n = 2 for other PEGs.</p

DMSO promotes spontaneous heme crystallization in acidic conditions.

<p>(A) Spontaneous heme crystallization was performed from a 100 µM solution at 27.7% v/v DMSO in 0.5 M sodium acetate buffer pH 4.8, over 24 h at 28°C. Data are expressed as mean ± SEM, of three different experiments. (B) The final reaction products were then characterized by FTIR spectroscopy. The large Nujol peaks in the region between 1320 cm⁻¹ and 1550 cm⁻¹ are depicted in light gray whereas the key βH peaks are shown at 1664 cm⁻¹ and 1210 cm⁻¹. (C) X-ray powder diffraction (XRD) was also used to confirm the identity of βH. (D) Scanning electron microscopy (SEM) was used to investigate the external morphology of the βH produced.</p

Reduction in water activity drives both heme solubility and βH formation under acidic conditions.

<p>Values of heme in solution were obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012694#pone-0012694-g001" target="_blank">Figure 1B</a> and values of βH produced was obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012694#pone-0012694-g002" target="_blank">Figure 2A</a>. Black square: nmols heme in solution; open circle: βH. Water activity was calculed based on values obtained in Dupont and Pougeois, 1983 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012694#pone.0012694-Dupont1" target="_blank">[43]</a>.</p

DMSO promotes spontaneous heme solubilization in acidic conditions.

<p>(A) Different concentrations of DMSO in 0,5 M sodium acetate buffer pH 4.8 and 100 µM heme with a final volume of 1.0 mL were shaken for 10 minutes and centrifuged at 10 000×g. for 10 min. The supernatants were analyzed by uv-visible spectroscopy between 300 nm and 800 nm. An expansion magnification of the dotted box is shown in the inset. Dashed line black: control; dashed line gray: 4.6% DMSO; pale gray: 8.3% DMSO; dark gray: 15.1% DMSO; black: 27.7% DMSO. (B) Heme content in solution was quantified using the alkaline pyridine method. Data are expressed as mean ± SEM, of three different experiments in B.</p

Scanning electron micrographs of βH induced by PEGs.

<p>Scanning electron microscopy (SEM) was used to investigate the external morphology of the βH crystals produced by different PEGs. Well formed crystals are seen in the presence of PEG 6.000, 8.000 and 20.000 which closely resemble hemozoin. Less regular crystals appear to be formed by PEG 3.350 and few if any are formed in the presence of PEG 300.</p

Fourier transformed infrared spectroscopy identifies the crystals produced by different lipids as β-hematin.

<p>The crystals were produced by 100 µM uPC (A), uPS (B), uPE (C) or 10 µg/mL total lipids isolated from PMVM of <i>R. prolixus</i> previously fed with plasma (D) or blood (E). The characteristic iron-carboxylate peaks of β-hematin at 1210 and 1663 cm⁻¹ are shown.</p

Structural and physico-chemical features of the glycerophospholipids investigated in the present work.

a<p>T_m values were obtained from the literature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088976#pone.0088976-Silvius1" target="_blank">[67]</a>.</p

Kinetics of heme crystallization promoted by different commercial and biological lipids.

<p>Heme crystallization reactions were induced <i>in vitro</i> mediated by uPC, uPS or uPE (100 µM), a blended phospholipid mixture of commercial uPS (14%), uPC (32%) and uPE (51%) or 10 µg/mL of total lipids isolated from PMVM of <i>R. prolixus</i> previously fed with plasma or blood. Data are expressed as mean ± SD, of at least three different experiments and fitted using the Avrami equation as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088976#s2" target="_blank">methods section</a>. To perform the Avrami analysis, the uPC-induced kinetics were independently analyzed at early and late times, which are shown as insets.</p

Unsaturated phosphatidylethanolamine produced homogeneous crystals morphologically similar to those induced by <i>R. prolixus</i> midgut lipids.

<p>Transmission electron microscopy of crystals induced by 100 µM uPC (A), uPS (B) or uPE (C) or 10 µg/mL total lipids isolated from <i>R. prolixus</i> midgut content previously fed with plasma (D) or blood (E). The inset shown in uPC represent a very small population of regularly shaped crystals produced by uPC. Scale bars represent 100 nm for all images, including the inset.</p