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A B S T R A C T Incubation of a 0.5% suspension of washed, normal mouse erythrocytes with ferriprotoporphyrin IX (FP) at 37°C and pH 7.4 caused potassium loss, swelling, increased susceptibility to hypotonic lysis, and finally hemolysis. Hemolysis was not inhibited by incubation in the dark, malonyldialdehyde was not produced, and various free radical scavengers had no effect on the hemolysis. Only the sulfhydryl compounds, cysteine, dithiothreitol, and mercaptoethanol protected erythrocytes from FP. Potassium loss reached 90% within 30 min of exposure to 5 uM FP. This amount of FP caused >50% hemolysis within 2.5 h. Sucrose (0.1 M) completely prevented hemolysis but had no effect on potassium loss. Likewise, reducing the temperature from 37 to 25°C greatly retarded hemolysis but had no effect on potassium loss. These observations indicate that FP impairs the erythrocyte's ability to maintain cation gradients and induces hemolysis by a colloid-osmotic mechanism.

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A B S T R A C T Incubation of a 0.5% suspension of washed normal mouse erythrocytes with ferriprotoporphyrin IX (FP) for 2.5 h at 37°C and pH 7.4 results in sufficient membrane damage to produce hemolysis. A sigmoidal dose-response curve is followed with 50% hemolysis being produced by 4 ,uM FP. Complete hemolysis is produced by 6 ,uM FP. The hemolytic process has at least two phases: a lag phase of -45 min, during which little hemolysis occurs, and a phase characterized by rapid hemolysis. Chloroquine, which binds tightly to FP, enhances the effect of FP by eliminating the lag phase. Under the conditions of these experiments, maximum enhancement is observed with chloroquine concentrations in the range of 5-25 ,uM. Since FP is produced when malaria parasites digest hemoglobin, it may mediate a chemotherapeutic effect of chloroquine by forming a complex with the drug that could enhance the toxicity of FP for biological membranes, including those of the parasite.

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Human erythrocytes were treated with menadione to oxidatively denature hemoglobin and release ferriprotoporphyrin IX (ferriheme, FP) intracellularly. The high affinity of FP for chloroquine was used to detect its release. After incubation for 1 hr at 37 degrees C and pH 7.4 with 0.5 mM menadione, erythrocytes bound 14C-chloroquine with an apparent dissociation constant of 10(-6)M. Untreated erythrocytes did not bind chloroquine with high affinity. At a chloroquine concentration in the medium of 2 microM, for example, menadione-treated erythrocytes bound 70 mumole chloroquine/kg and untreated erythrocytes bound 13.4 mumole/kg. The intracellular location of FP released by menadione was verified by finding that Tween 80 did not prevent chloroquine binding. By contrast, Tween 80 inhibited the binding of chloroquine to erythrocytes treated with extracellular FP. The hemolytic response to menadione was characteristic of the hemolytic response to FP. Thus, 5 microM chloroquine caused hemolysis to increase to 60% from baseline values of 5% in experiments using erythrocytes treated either with 0.5 mM menadione or with 5 microM FP; and, in both cases, the potentiating effect of chloroquine was inhibited by 1 microM mefloquine or 10 microM quinine. Higher concentrations of menadione caused hemolysis in the absence of chloroquine. We conclude that FP released by menadione exists intracellularly in a form that is accessible to bind chloroquine and to express its lytic activity.