Figure 5

Protein expression of antioxidant enzymes in early trophozoite stage parasites growing in normal and oxidatively stressed RBCs. Early trophozoite stages were isolated from cultured synchronized parasites. Parasites were isolated from host RBCs by Sendai-virus treatment and lysed by freeze-thawing and sonication. Ten to 15 μg of protein extract of control (Column A) and oxidatively stressed (Column B) trophozoite stages were separated by SDS-PAGE and blotted onto nitrocellulose. The blots were hybridized with polyclonal antibodies directed against parasite superoxide dismutases (SOD-1 and SOD-2), thioredoxin reductase (Trx), thioredoxin-dependent peroxidases (1-Cys-Prx, 2-Cys-Prx), and glutathione reductase (GR), and subsequently with the secondary anti-rabbit or rat horseradish-peroxidase-coupled antibody and the bands detected by chemiluminescence and densitometric quantification. Column C shows protein expression increases in oxidatively stressed cells vs untreated controls, expressed as -fold variation of band densitometry. The results shown are representative from 1–3 independent experiments. For details, see Methods.