Hemozoin: a case of heme crystal engineering

Abstract

During the pathogenic blood stage of a malaria infection, the Plasmodium parasites degrade hemoglobin as a source of nutrition. As a consequence, free heme, known to be toxic to the parasite, is released. It is believed that the parasite circumvents heme toxicity by sequestering the heme molecules into a dark brown crystalline material known as hemozoin. The molecular details associated with the formation of hemozoin and its synthetic counterpart, beta-hematin, are presented in this dissertation. Firstly, the biological mediator of hemozoin formation was investigated. Neutral lipid droplets (NLDs) were shown to be sufficient at mediating the production of brown pigments that are morphologically and chemically identical to hemozoin. Optimal partitioning of heme into NLDs was pH dependent with maximal heme conversion at a pH condition similar to that of the parasite’s digestive food vacuole, the biological site of crystallization. The rate of beta-hematin formation was rapid enough to protect the parasite from heme toxicity. Secondly, the interfacial interactions between lipid molecules and heme were investigated using Langmuir-Blodgett monolayer creation techniques. Comparisons of these surface pressure-area isotherms revealed that the biological composition of neutral lipid is characterized by disordered packing of lipids. This fluid lipid surface may account for the low activation energy measured for beta-hematin formation associated with NLDs. Substituted protoporphyrin IX compression studies suggest that hemozoin nucleation begins when the propionic group of a heme unit anchors to the polar head group of the lipid molecules. Thirdly, crystallization parameters associated with beta-hematin formation was examined using various solvent conditions to facilitate heme solubility. The formation of beta-hematin using the aprotic solvent dimethylsulfoxide and some polyethyleneglycols demonstrates that crystallization is accelerated by increasing heme solubilization in acidic conditions, resulting on increased dispersion of amorphous heme precipitates. Crystallization data support the notion that modulation of the water activity is important mechanism to support spontaneous heme crystallization. Futhermore, through proper manipulation of solvent properties, the morphologies of beta-hematin can be controlled. Finally, beta-hematin crystals were applied to phage display technologies to identify short peptide sequences that specifically recognize select crystal face. Isolated peptides were sufficient at mediating beta-hematin formation.