2.4. Inhibition of β-hematin formation assay

The assay was performed using a lipid as a catalyst to promote crystallization (Pisciotta et al., 2007; Fitch et al., 1999). Briefly, drug stock solutions were prepared in DMSO and were used at a final concentration of up to 30 mM. A heme stock (10 mM) was made in DMSO and was diluted to 50 μM with 100 mM sodium acetate (pH 4.8). A 10 mM 1-monooleoyl-rac-glycerol (MOG) stock was made in ethanol and was sonicated before a 50 μM heme stock was added to make 25 μM MOG–50 μM heme in 100 mM sodium acetate (pH 4.8). The 25 μM MOG–50 μM heme solution was sonicated and added to the assay plate at 100 μL/well. The plates were incubated at 37 °C for 2 h to allow crystallization, followed by the addition of 100 μL of 200 mM sodium bicarbonate (pH 9.1) to solubilize any remaining monomeric heme. After incubation for 30 min at room temperature, the amount of solubilized monomeric heme was determined by measuring the absorbance at 405 nm. Finally, 20 μL of 1 M sodium hydroxide were added to the plates to dissolve any crystals that had been formed. The absorbance was read at 405 nm to determine the total amount of heme present in each well. The inhibition of heme crystallization was determined as a function of the amount of monomeric heme that was not crystallized divided by the total amount of heme present in the assay mixture. The results are expressed as IC₅₀ values based on the percentage inhibition of β-hematin formation by the compounds GIQ, CEQ, PCQ and DAQ. Each test was performed in triplicate in at least two different experiments.