Background

Heme, an organic molecule, plays a vital role in virtually all living organisms. For example, heme binds to hemoglobin and myoglobin for oxygen transport and serves as a co-factor for several enzymes within the electron transport chain for mitochondrial respiration (Ponka, 1999). Previous literature has shown that Toxoplasma has a fully functional de novo heme biosynthesis pathway (Bergmann et al., 2020). Genetic deletion of Toxoplasma heme biosynthetic enzymes results in decreased replication in vitro (Bergmann et al., 2020; Krishnan et al., 2020; Tjhin et al., 2020) and the loss of acute virulence in a murine model (Bergmann et al., 2020); therefore, inhibition of de novo heme production sheds light on the development of novel therapeutic strategies for managing Toxoplasma infections.

Generally, there are four methods for the measurement of heme abundance in cells: 1) Pyridine hemochrome-based heme quantitation (Sinclair et al., 2001). This strategy replaces the nitrogen ligands of protein-bound heme with pyridine under alkaline conditions. The resulting hemochrome is further reduced and oxidized before spectrophotometric quantitation; 2) Reversed-phase HPLC-based quantitation of heme and its intermediates (Sinclair et al., 2001). An acetone/HCl/water solution is used to extract heme and its biosynthetic intermediates from intact cells or cell homogenates, followed by separation on a C18 HPLC column. The standards of pure heme and its intermediates are run on the column before sample measurement to help recognize their peaks and quantitate their abundances; 3) Protoporphyrin IX (PPIX)-based fluorescence assay (Sinclair et al., 2001). PPIX is produced by the penultimate enzyme within the classic de novo heme biosynthesis pathway (Phillips, 2019). PPIX is further conjugated with a ferrous iron group to form a functional heme molecule. The non-conjugated PPIX molecule displays strong fluorescence, whereas heme is non-fluorescent; therefore, stripping ferrous ion from heme generates fluorescent PPIX, which can be quantitated by a fluorometer and represents heme abundance; 4) Biosensor-based heme quantitation by live-cell imaging or flow cytometry (Song et al., 2015; Hanna et al., 2016; Yuan et al., 2016). Genetically encoded hemoproteins, such as horseradish peroxidase (HRP)- or ascorbate peroxidase (APX)-based biosensors, can be expressed in different organelles to detect their labile heme content (Yuan et al., 2016). Additionally, the heme-binding proteins are genetically fused to heme-sensitive fluorescent proteins to quantitate labile heme within live cells by ratiometric fluorescence quantitation or fluorescence resonance energy transfer (FRET) (Song et al., 2015; Hanna et al., 2016).

The pyridine hemochrome-based strategy is not very sensitive and requires large amounts of parasites as initial material. The HPLC-derived quantitation requires expensive equipment and columns. Additionally, it only can quantitate one sample per run. The biosensor-based method demands the implementation of biosensors in target organisms. Overexpression of biosensors may disturb labile heme pools in the cells and further impair their health status. The PPIX-based fluorescence heme quantitation is economical and sensitive. Moreover, it can be performed in 96-well plate format as a semi high-throughput strategy for multiple samples simultaneously.

In the procedure for the PPIX-based heme quantitation assay, the heme-bound ferrous ion can be chemically removed by oxalic acid via boiling before measurement. During the assay, non-boiled parallel samples must be included to detect background fluorescence signals within parasite strains, which will be subtracted from the corresponding boiled samples. Here, we modify the previously published PPIX-based fluorescence heme quantitation assay (Sinclair et al., 2001). In this protocol, the parasites are mechanically liberated from host cells, filter-purified, and ruptured by sonication prior to heme quantitation. As an example, we measured and compared heme abundances in wildtype parasites and a heme-deficient transgenic Toxoplasma strain. This method employs a plate reader to quantitate heme in a 96-well plate format; it shows high sensitivity and only requires 5-10 × 10⁷Toxoplasma parasites for measurement, which can be harvested from 1-2 T25 flasks. This strategy can be modified for measuring heme abundances in other apicomplexan parasites.