Fig. 2

Flow diagram showing the haem fractionation assay: original flask method (left) versus increased throughput plate method (right). The haem fractionation assay is a cellular fractionation technique based on the ability of neutral aqueous pyridine to selectively form a low spin haem-pyridine complex with free haem in the presence of Hz [13]. The original method was performed in 250 ml culture flasks, testing a single drug concentration at a time and performing spectroscopic measurements in a cuvette [12]. The modified method was performed in 24-well plates, testing several drug concentrations, four replicates at a time and using a multiwell plate reader to record absorbance, resulting in a six-fold increase in output. Immature ring stage parasitized red blood cells were inoculated with several different increasing drug concentrations. After 32 h, mature trophozoites were harvested via saponin lysis of erythrocytes. Following hypotonic lysis and centrifugation, SDS treated soluble Hb was measured in the supernatant as a low spin haem-pyridine complex. The pellet was further treated with SDS and pyridine to solubilize free haem, measured in the supernatant following centrifugation. Hz was measured after solubilizing the pellet in NaOH, neutralizing with HCl and treating with pyridine