An assay procedure to investigate the transformation of toxic heme into inert hemozoin via plasmodial heme detoxification protein

Most antimalarial therapeutics, including chloroquine and artemisinin, induce free heme-mediated toxicity in Plasmodium. This cytotoxic heme is produced as a by-product during the large-scale digestion of host hemoglobin. Conversion of this host-derived heme into inert crystalline hemozoin is the only defense mechanism in Plasmodium against heme-induced cytotoxicity. Heme detoxification protein (HDP), a highly conserved plasmodial protein, is reported to be the most efficient biological mediator for heme to hemozoin transformation. Despite its significance, HDP has never been extensively studied for heme transformation into hemozoin. Therefore, we wish to develop a method to study the HDP-mediated transformation of heme into hemozoin. We have adopted, modified, and optimized the pyridine hemochrome assay to study HDP catalysis and use substrate and time kinetics to study the HDP-mediated transformation of heme into hemozoin. In contrast to the previously reported assay for HDP, we found that the new assay is more precise, accurate, and handy, making it more suitable for kinetic studies. HDP-mediated transformation of heme into hemozoin is not a single-step process, and involves a transient intermediate, most likely a cyclic heme dimer. The kinetics and the manner of HDP-mediated hemozoin production are dependent on the substrate concentration, and a small fraction of substrate remains untransformed to hemozoin irrespective of reaction time. Combining HDP as a catalyst and the pyridine hemochrome assay will facilitate the efficient screening of future antimalarials.