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Approximately 70% of the initial ferriprotoporphyrin IX polymerizing activity in cell-free preparations of erythrocytes infected with Plasmodium berghei was recovered in a chloroform extract. No polymerizing activity remained in the residue. In studies to identify substances that promote FP polymerization, arachidonic, linoleic, oleic, and palmitoleic acids, 1-mono- and di-oleoylglycerol, and the detergents, SDS, Tween 80, and n-octyl-glucopyranoside, were active. Tri-oleoylglycerol, cholesterol, di-oleoylphosphatidylethanolamine, and stearic and palmitic acids were inactive. The model lipid, mono-oleoylglycerol (250 nmol), co-precipitated with FP from a 0.09 M acetate medium at pH 5 and promoted the polymerization of 215 nmol (61%) of the ferriprotoporphyrin IX in the precipitate during a 24-h incubation at 37 degrees C. Polymerization was maximal at pH 5, it was approximately linear for 2 h, and it continued at a decreasing rate for 24 h. The polymer contained exclusively ferriprotoporphyrin IX (97+/-1.3%, mean+/-S.E., n=4) and exhibited the solubility and the electronic absorption and infrared spectral characteristics of the sequestered ferriprotoporphyrin IX of hemozoin. Detergents presumably promote polymerization in an acid medium by helping to dissolve monomeric FP. We suggest that unsaturated lipids co-precipitate with FP in the parasite's acidic food vacuole and also dissolve sufficient monomeric FP to allow polymerization.

Erythrocytes infected with chloroquine-sensitive Plasmodium falciparum bind chloroquine with an apparent intrinsic association constant of 1.5 x 10(7) liters per mole. Such high-affinity binding of chloroquine is absent or deficient in uninfected erythrocytes and in erythrocytes infected with chloroquine-resistant Plasmodium falciparum.

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.

Malaria parasites isolated from mouse erythrocytes are lysed by ferriprotoporphyrin IX chloride (hemin) or by a chloroquine-hemin complex in amounts that could be produced by release of less than 0.1 percent of the heme in erythrocytic hemoglobin. This effect of hemin may explain the protection against malaria provided by thalassemia and other conditions causing intracellular denaturation of hemoglobin. The toxicity of the chloroquine-hemin complex may explain the selective antimalarial action of chloroquine.

Abstract.-Chloroquine-14C was used to study the processes which concentrate chloroquine in mouse red blood cells infected with chloroquine-sensitive or with chloroquine-resistant Plasmodium berghei. The initial rates of uptake and exchange of chloroquine-14C were both too fast to measure, yet large concentration gradients were maintained by the cells. When red blood cells were exposed to 10-8 M chloroquine at 220C, with pH between 7.2 and 7.4, steady-state gradients of chloroquine-14C were approximately 600: 1 (cells: medium) for cells infected with chloroquine-sensitive parasites, 100:1 for cells comparably infected with chloroquine-resistant parasites, and 14: 1 for uninfected cells. The processes responsible for these gradients were saturable, in agreement with the proposal of chloroquine binding to cellular constituents. No degradation of chloroquine was detected.The major difference between the chloroquine-sensitive and -resistant parasites was a deficiency of high-affinity binding of chloroquine by cells infected with chloroquine-resistant parasites. This deficiency explains the reduced ability of chloroquine-resistant parasites to concentrate chloroquine, and it suggests that chloroquine resistance is due to a decrease in the number, affinity, or accessibility of chloroquine receptor sites on a constituent of the malaria parasite.Previous work has demonstrated that mouse red blood cells infected with chloroquine-sensitive Plasmodium berghei can achieve concentration gradients of chloroquinel of 100: 1 (cells: plasma) in vivo, whereas cells infected with a chloroquine-resistant variant of this same strain of P. berghei concentrate chloroquine less than half as well.2' 3 Since the partial loss of ability to concentrate chloroquine is associated with chloroquine resistance in this model system, the present work was undertaken to characterize and to compare the concentrative processes of chloroquine-sensitive and -resistant P. berghei.Materials and Methods.-Infections were maintained in mice by weekly passage of chloroquine-sensitive P. berghei and biweekly passage of chloroquine-resistant P. berghei.4Both types of parasites were descendants of the same parent strain of P. berghei, the NYU-2 strain; the chloroquine-resistant variant was the one previously used in studies of the processes which concentrate chloroquine in infected red blood cells in vivo.2, 8 To pass the infections, normal mice were each given an intraperitoneal injection of 0.1 ml of a 1: 25 dilution of blood which had 30-90% of its red blood cells parasitized. The blood used for passage of the chloroquine-resistant parasites was always obtained from mice each one of which was treated daily with 1 mg of chloroquine intraperitoneally for 4 days of each week after inoculation with parasitized blood. This amount of chloroquine did not prevent the fatal progression of malaria caused by chloroquine-resistant parasites although it promptly cures mice infected with chloroquine-sensitive parasites.8 Under our conditions, the chloroquine-sensitive para...

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