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

Abstract

BackgroundHemozoin (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 FindingsWe 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.ConclusionsThe 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.

Phospholipid Membrane-Mediated Hemozoin Formation: The Effects of Physical Properties and Evidence of Membrane Surrounding Hemozoin

“…The differences in morphology between dioleoyl-PC and dipalmitoyl-PC vesicles were probably due to their different physical properties, in particular the different states – fluid or gel – caused by the differences in T m values. Moreover, since the hydrophobic interactions between their inducers and heme have been proposed as an important force in the creation of a precursor heme dimer [38], [45], [54], [55], it was interesting to explore the correlation between the physical properties of phospholipids and their ability to induce BH. Our results demonstrated that phospholipids could induce BH formation only in the fluid phase, and could not do so in the gel phase.…”

The free heme concentration in healthy human erythrocytes

“…The highest absorbance of such solutions, in the Soret band near 400 nm, is below 0.1. Hence, spectroscopic determination of concentrations below this limit is prone to high error [25]. …”

Biomimetic Approaches to Understanding the Mechanism of Haemozoin Formation

“…The energy barrier to crystal nucleus formation is known to depend on surface tension (Myerson, 1993), so the authors proposed that alcohols also play a role in reducing the energy barrier to crystallisation in this way. A recent study on -haematin formation in aqueous DMSO and solutions of several polyethylene glycols (PEGs) has provided direct support for the solubilisation hypothesis (Stiebler et al, 2010b). In this investigation it was demonstrated spectrophotometrically that Fe(III)PPIX solubility at pH 4.8 in 0.5 M acetate buffer increases linearly with increasing DMSO concentration and that the yield of -haematin is directly proportional to the solubility of Fe(III)PPIX in this medium.…”