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Abstract

The acidic pH of the digestive vacuole (DV) of P. falciparum is thought to play a major role in the mechanisms of digestion of host erythrocyte hemoglobin, detoxification of heme, and chloroquine (CQ) action and resistance. However, a definitive measurement of the DV pH has been technically difficult. A previous study by Abu Bakar (2015) measured the DV pH by using fluorescein isothiocyanate-dextran (FITCdextran), a ratiometric pH indicator loaded into the malaria parasite"s DV that had been isolated using saponin (DV sap). To validate the FITCdextran response to the DV sap pH, pH of the DV isolated by digitonin permeabilization (DV digi) was measured. The DV digi pH of the CQsensitive parasite (D10) was 5.66 ± 0.07 that is approximately similar to that of the DV sap pH (5.27 ± 0.03) estimated using the same probe. The DV digi pH of the CQ-resistant parasite (Dd2) (5.62 ± 0.12) was not significantly different from the CQ-sensitive parasite"s DV digi pH (P> 0.3). Re-acidification of the DV digi was also observed upon the addition of 2 mM ATP to the ATP-depleted medium. These data validate the use of FITC-dextran for quantitative DV pH analysis of the parasite isolated using either digitonin or saponin. INTRODUCTION: The potential importance of the malaria parasite"s DV pH in the degradation of hemoglobin and detoxification of heme to harmless hemozoin has long been discussed 1, 2, 3. A study by Chugh et al. reported the presence of a collection of proteins in the DV that is required for the underlying mechanisms of hemoglobin digestion and hemozoin formation. This protein complex comprised parasite proteases such as histo aspartic protease, falciparum 2/2', and plasmepsin II and IV.

Ellagic Acid Induces in vitro Alkalinisation of the Digestive Vacuole in Drug-Sensitive Plasmodium falciparum Strain

“…The pH calibration curve of FITC-dextran was generated to measure pH of the digestive vacuole ( 28 , 42 ). Resealed erythrocytes containing FITC-dextran (2% haematocrit) were suspended in different buffers (20 mM) (MES, pH 4.0, 5.5 and 6.0; NaH 2 PO 4 , pH 6.5, 7.0, 7.5 and 8.0; TRIS, pH 9.0) supplemented with 150 mM NaCl and in the presence of 10 μM ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP; Sigma-Aldrich, USA).…”