Accelerated denaturation of hemoglobin and the antimalarial action of chloroquine

Abstract

To study the antimalarial action of chloroquine, normal mouse erythrocytes were used as surrogates for erythrocytoid bodies. These bodies form in the endosomes of intraerythrocytic malaria parasites as they feed on their host and consist of erythrocyte cytoplasm enclosed in a vestige of the erythrocyte membrane. In suspensions of normal erythrocytes or lysates (equivalent to 5 microl of erythrocytes per ml in each case), hemoglobin underwent denaturation when it was incubated at 38 degrees C in 150 mM sodium acetate (pH 5). It is reasonable to assume that the same phenomenon occurs in acidic endosomes. Addition of 100 microM chloroquine to the incubation mixture caused the rate of hemoglobin denaturation to double to 40 nanomoles per hour per ml of packed erythrocytes. This effect required the presence of erythrocyte stroma and was inhibited by reducing the temperature to 24 degrees C or increasing the pH to 6. We propose that the primary antimalarial action of chloroquine is to bind to ferriprotoporphyrin IX (FP) and remove it from oxidized hemoglobin, thus producing toxic FP-chloroquine complexes and an excess of denatured globin. Furthermore, we suggest that these substances inhibit endosomal maturation and thereby cause hemoglobin accumulation in immature endosomes and masking of the lipids needed for FP dimerization. The term "masking" is used to signify that unsaturated lipids are present in parasitized erythrocytes but are specifically unavailable to promote FP dimerization.

FIG. 1.

Effect of chloroquine on hemoglobin denaturation. Suspensions of intact erythrocytes or lysates, each prepared from 50 μl of packed erythrocytes, were diluted to 10 ml with 150 mM sodium acetate (pH 5) and incubated for 1 h at 38°C under room air in the presence or the absence of chloroquine. Shaded bars indicate the presence of 500 μM chloroquine. Means ± standard deviations for five experiments are shown. For each preparation, the effect of chloroquine was significant (P < 0.001, t test). The differences between intact erythrocytes and lysates were not statistically significant. The data are expressed as the nanomoles of denatured hemoglobin minus the background per ml of packed erythrocytes per hour. The background value was 24 ± 13 nanomoles of denatured hemoglobin per ml of packed erythrocytes for this set of experiments.

FIG. 2.

Effect of erythrocyte stroma on hemoglobin denaturation. Aliquots of erythrocyte cytoplasm, each derived from 50 μl of packed erythrocytes, and various amounts of erythrocyte stroma were diluted to 10 ml with 150 mM sodium acetate (pH 5) and incubated for 1 h at 38°C under room air. The ordinate shows the nanomoles of denatured hemoglobin minus the background per ml of packed erythrocytes per hour, and the abscissa shows the amounts of stroma derived from various volumes of erythrocytes (expressed in terms of μl of packed erythrocytes). Solid symbols indicate the presence of 100 μM chloroquine. Means ± standard deviations are shown for four experiments except for zero time in the presence of 100 μM chloroquine, when there were only three experiments.

FIG. 3.

Relationship between chloroquine concentration and hemoglobin denaturation. Suspensions of lysates, each prepared from 50 μl of packed erythrocytes, were diluted to 10 ml with 150 mM sodium acetate (pH 5) and incubated for 1 h at 38°C under room air in the absence of chloroquine or in the presence of various concentrations of chloroquine. The nanomoles of denatured hemoglobin produced per ml of packed erythrocytes per hour in the presence of chloroquine minus the amount produced in the absence of chloroquine are shown. In the absence of chloroquine, 52 ± 12 nanomoles of denatured hemoglobin was produced per ml of packed erythrocytes per hour. Means ± standard deviations and the number of experiments performed at each concentration of chloroquine (in parentheses) are shown.

FIG. 4.

Effect of pH and temperature on hemoglobin denaturation. (Lower panel) Effect of pH. Suspensions of erythrocyte lysates, each prepared from 50 μl of packed erythrocytes, were diluted to 10 ml with 150 mM sodium acetate at pH 5 or 6 or with the standard medium at pH 7.4 and incubated for 1 h at 38°C under room air. Shaded bars indicate the presence of 100 μM chloroquine. The data are expressed as nanomoles of denatured hemoglobin minus the background per ml of packed erythrocytes per hour. Means ± standard deviations are shown for four experiments. (Upper panel) Effect of temperature. Suspensions of erythrocyte lysates, each prepared from 50 μl of packed erythrocytes, were diluted to 10 ml with 150 mM sodium acetate (pH 5) and incubated under room air for 1 h at the indicated temperatures. Shaded bars indicate the presence of 100 μM chloroquine. The data are expressed as nanomoles of denatured hemoglobin minus the background per ml of packed erythrocytes per hour. Means ± standard deviations for four experiments are shown.

FIG. 5.

Time course of the effect of chloroquine on hemoglobin denaturation. Suspensions of erythrocyte lysates, each prepared from 50 μl of packed erythrocytes, were diluted to 10 ml with 150 mM sodium acetate (pH 5) and incubated for various lengths of time at 38°C under room air in the presence or the absence of 100 μM chloroquine. The nanomoles of denatured hemoglobin minus the background per ml of packed erythrocytes are shown for each time interval. Solid symbols indicate the presence of 100 μM chloroquine. The dashed line shows the difference between incubations in the presence and the absence of chloroquine. Means ± standard deviations are shown for four experiments.