Hemozoin-free Plasmodium falciparum mitochondria for physiological and drug susceptibility studies

Isolation of mitochondria of high purity and with intact enzymatic activities from malaria parasites has proven to be a major obstacle in characterizing the parasite mitochondrial physiology. We describe here an improved procedure for the isolation of a mitochondrially enriched preparation from the trophozoite stage of erythrocytic Plasmodium falciparum, combining disruption by N2 cavitation and differential centrifugation with magnetic removal of hemozoin-associated material. These mitochondrial preparations may be used to assay various mitochondrial enzyme activities, such as succinate and dihydroorotate dehydrogenases, ubiquinol-cytochrome c oxidoreductase, and cytochrome c oxidase. They also exhibit a low level of ATPase activity, which is only marginally inhibited by classical inhibitors. We have used this preparation to determine the susceptibility of mitochondrial activities to drugs and drug candidate compounds in both “wild type” and transgenic parasites.

We developed a mitochondrial preparation procedure incorporating a magnetic separation step to remove nearly all hemozoin. Use of this preparation in spectrophotometric measurements is illustrated.201Research highlights▶ Plasmodium falciparum mitochondria contain established and emerging drug targets. ▶ Parasite physical characteristics and hemozoin impede the isolation of mitochondria. ▶ A magnetic separation step during isolation largely removes hemozoin. ▶ This preparation facilitates spectrophotometric assays of mitochondrial enzymes.