Figure 2

Haem quantification by different techniques. Different concentrations of haematin (10 μM – 1.2 nM) were used to compare the sensitivity of previously described techniques to quantify haem in a 96-well based format. In panel A, haematin was measured with a spectrophotometer at 405 nm (n = 4 for each concentration). Panel B shows the measurements of haematin concentrations by haem-enhanced luminescence with the same reagent conditions as described by Schwarzer and colleagues (20) for a cuvette-based system. Background absorbance/luminescence from a blank sample was subtracted from all the measurements. The adjusted chemo-luminescence protocol (100-fold higher luminol and peroxide concentrations buffered around pH 10.4) was used to measure the same concentrations of haematin in panel C (n = 8 for each concentration in B and C). The horizontal dashed line denotes the accuracy limit of the assay. The time-dependence of the luminescence signal and the effect of 2% SDS are displayed in panel D and E, respectively. The arrow in panel D denotes the start of the kinetic reading after an initial delay of twelve seconds, and the vertical dashed line indicates the moment of luminescence detection during the experimental readings. In panel F, natural Hz was isolated from trophozoites (troph) and livers, its concentration was determined by absorption at 405 nm and the concentration-dependence of the haem-enhanced luminescence was measured. Inset of panel F is a picture demonstrating the birefringence of the isolated Hz. The amount of Hz/trophozoite was calculated and the corresponding number of trophozoites (# troph) is integrated in the figure as a second X-axis