Increase on the Initial Soluble Heme Levels in Acidic Conditions Is an Important Mechanism for Spontaneous Heme Crystallization In Vitro

BACKGROUND: Hemozoin (Hz) is a heme crystal that represents a vital pathway for heme disposal in several blood-feeding organisms. Recent evidence demonstrated that β-hematin (βH) (the synthetic counterpart of Hz) formation occurs under physiological conditions near synthetic or biological hydrophilic-hydrophobic interfaces. This seems to require a heme dimer acting as a precursor of Hz crystals that would be formed spontaneously in the absence of the competing water molecules bound to the heme iron. Here, we aimed to investigate the role of medium polarity on spontaneous βH formation in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the effect of water content on spontaneous βH formation by using the aprotic solvent dimethylsulfoxide (DMSO) and a series of polyethyleneglycols (PEGs). We observed that both DMSO and PEGs (3.350, 6.000, 8.000, and 22.000) increased the levels of soluble heme under acidic conditions. These compounds were able to stimulate the production of βH crystals in the absence of any biological sample. Interestingly, the effects of DMSO and PEGs on βH formation were positively correlated with their capacity to promote previous heme solubilization in acidic conditions. Curiously, a short chain polyethyleneglycol (PEG 300) caused a significant reduction in both soluble heme levels and βH formation. Finally, both heme solubilization and βH formation strongly correlated with reduced medium water activity provided by increased DMSO concentrations. CONCLUSIONS: The data presented here support the notion that reduction of the water activity is an important mechanism to support spontaneous heme crystallization, which depends on the previous increase of soluble heme levels

Unsaturated glycerophospholipids mediate heme crystallization: biological implications for hemozoin formation in the kissing bug Rhodnius prolixus

Hemozoin (Hz) is a heme crystal produced by some blood-feeding organisms, as an efficient way to detoxify heme derived from hemoglobin digestion. In the triatomine insect Rhodnius prolixus , Hz is essentially produced by midgut extracellular phospholipid membranes known as perimicrovillar membranes (PMVM). Here, we investigated the role of commercial glycerophospholipids containing serine, choline and ethanolamine as headgroups and R. prolixus midgut lipids (RML) in heme crystallization. All commercial unsaturated forms of phospholipids, as well as RML, mediated fast and efficient β-hematin formation by means of two kinetically distinct mechanisms: an early and fast component, followed by a late and slow one. The fastest reactions observed were induced by unsaturated forms of phosphatidylethanolamine (uPE) and phosphatidylcholine (uPC), with half-lives of 0.04 and 0.7 minutes, respectively. β-hematin crystal morphologies were strikingly distinct among groups, with uPE producing homogeneous regular brick-shaped crystals. Interestingly, uPC-mediated reactions resulted in two morphologically distinct crystal populations: one less representative group of regular crystals, resembling those induced by uPE, and the other largely represented by crystals with numerous sharp edges and tapered ends. Heme crystallization reactions induced by RML were efficient, with a heme to β-hematin conversion rate higher than 70%, but clearly slower (t1/2 of 9.9-17.7 minutes) than those induced by uPC and uPE. Interestingly, crystals produced by RML were homogeneous in shape and quite similar to those mediated by uPE. Thus, β-hematin formation can be rapidly and efficiently induced by unsaturated glycerophospholipids, particularly uPE and uPC, and may play a role on biological heme crystallization in R. prolixus midgut

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

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

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

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

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