Intracellular ferriprotoporphyrin IX is a lytic agent

Abstract

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.

Regulation of heme polymerizing activity and the antimalarial action of chloroquine

“…The rationale is as follows. Chloroquine acts on the negative arm of the regulatory process to cause a loss of HPA I, and consequently unpolymerized FP accumulates and, through its membrane toxicity (4,11), causes autophagic vacuole formation. In addition, diverse metabolic inhibitors disrupt the negative arm of the regulatory process and thereby antagonize the effect of chloroquine.…”

Schistosomiasis Chemotherapy

“…Studies reported in 1972 indicated that the pigments formed during hemoglobin digestion by malarial and schistosomal parasites are “very similar if not identical” 47. After many controversies about the structure of malaria pigment, it is now considered that purified hemozoin of Plasmodium consists of ferriprotoporphyrin IX without any other components, such as proteins or fatty acids,48 and is identical to synthetic crystalline β‐hematin 49. More recently, the heme‐polymer pigment produced by S. mansoni was compared to β‐hematin and to pigments produced by other blood‐feeding organisms, such as Plasmodium , Haemoproteus , and Rhodnius 47.…”

Hemozoin: Oil versus water

“…Less than 10-20 μM heme can inhibit many Plasmodium enzymes in the digestive vacuole like plasmepsins and falcipains [11,12] and also enzymes in cytosol like glycolytic glyceraldehyde-3-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase [13,14]. Integration of the lipophilic heme molecule into biological membranes diminishes erythrocytes deformability and induces hemolysis [15][16][17][18]. Membrane heme also weakens the lipid bilayer by making it more susceptible to H 2 O 2 mediated lysis [16,18].…”