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An Iron-Carboxylate Bond Links the Heme Units of Malaria Pigment (Plasmodium/Hemoglobin/Hemozoin/Extended X-Ray Absorption Fine Structure) ANDREW F

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An Iron-Carboxylate Bond Links the Heme Units of Malaria Pigment (Plasmodium/Hemoglobin/Hemozoin/Extended X-Ray Absorption Fine Structure) ANDREW F Proc. Nati. Acad. Sci. USA Vol. 88, pp. 325-329, January 1991 Biochemistry An iron-carboxylate bond links the heme units of malaria pigment (Plasmodium/hemoglobin/hemozoin/extended x-ray absorption fine structure) ANDREW F. G. SLATER*t, WILLIAM J. SWIGGARD*, BRIAN R. ORTONf, WILLIAM D. FLITTER§, DANIEL E. GOLDBERG*, ANTHONY CERAMI*, AND GRAEME B. HENDERSON*¶ *Laboratory of Medical Biochemistry, The Rockefeller University, New York, NY 10021-6399; and tDepartment of Physics, and §Department of Biology and Biochemistry, Brunel University, Uxbridge, United Kingdom Communicated by Maclyn McCarty, October 15, 1990 (receivedfor review August 17, 1990) ABSTRACT The intraerythrocytic malaria parasite uses the purified pigment are shown to be identical to those of hemoglobin as a major nutrient source. Digestion of hemoglo- hemozoin in situ. Using chemical synthesis and IR, ESR, and bin releases heme, which the parasite converts into an insoluble x-ray absorption spectroscopy we demonstrate that hemo- microcrystalline material called hemozoin or malaria pigment. zoin consists of heme moieties linked by a bond between the We have purified hemozoin from the human malaria organism ferric ion of one heme and a carboxylate side-group oxygen Plasmodium falkiparum and have used infrared spectroscopy, of another. This linkage allows the heme units released by x-ray absorption spectroscopy, and chemical synthesis to de- hemoglobin breakdown to aggregate into an ordered insolu- termine its structure. The molecule consists of an unusual ble product and represents a novel way for the parasite to polymer of hemes linked between the central ferric ion of one avoid the toxicity associated with soluble hematin. heme and a carboxylate side-group oxygen of another. The hemes are sequestered via this linkage into an insoluble prod- MATERIALS AND METHODS uct, providing a unique way for the malaria parasite to avoid the toxicity associated with soluble heme. Materials. Hematin, hemin (ferriprotoporphyrin IX chlo- ride), dimethyl sulfoxide, pyridine, and KBr were obtained The dark brown discoloration of the liver, spleen, and brain from Aldrich. Sodium [1,2-14C]acetate was supplied by NEN. observed in patients suffering a severe malaria infection was All other chemicals and reagents were purchased from first reported by Lancisi in 1717 (1), long before the parasite Sigma. Elemental analyses were performed by Schwarzkopf itself was described. The crystalline substance causing this Microanalytical Laboratory (Woodside, NY). discoloration (called malaria pigment or hemozoin) is formed Parasite Culture. P.falciparum clone HB-3 was cultured in within the food vacuoles of intraerythrocytic malaria para- A+ human erythrocytes by the method ofTrager and Jensen sites as a product of the catabolism of hemoglobin (2). (11). Synchrony was maintained by sorbitol treatment, and Proteolysis of hemoglobin releases heme, which when solu- late trophozoite-stage parasites were harvested by saponin ble is highly toxic to biological membranes (3). As malaria lysis. parasites lack heme oxygenase, they are unable to cleave Hemozoin Purification. A crude extract of hemozoin was heme into an open-chain tetrapyrrole (4), and it is not prepared as described (8). In all subsequent steps, hemozoin excreted from the cell. Instead heme is detoxified by con- was suspended into buffer by brief sonication and then version into hemozoin, a process unique to the malaria removed from solubilized contaminants by centrifugation at organism. Hemozoin is released along with the merozoites 25,000 x g for 30 min at 4°C. To solubilize any contaminating when the infected erythrocytes burst and is scavenged by membranes, crude hemozoin was extracted twice for 3 hr at macrophages. This pigment is insoluble under physiologic room temperature in buffer (50 mM Tris-HCI, pH 7.4) con- conditions and remains undegraded within tissue macro- taining 2% sodium dodecyl sulfate. The hemozoin pellet was phages of the host for an extended period of time (5). washed three times in buffer, and residual proteins were The chemical nature of hemozoin has been the subject of removed by an overnight digestion in buffer containing study and speculation since Brown (6) first remarked on the proteinase E at 1 mg/ml. Insoluble material was recovered similarity between hemozoin and hematin (ferriprotoporphy- and washed as above, before being extracted in aqueous 6 M rin IX hydroxide). However, hemozoin is not identical to urea for 3 hr at 4°C. The purified hemozoin was pelleted by hematin, as hematin dissolves rapidly in solvents in which centrifugation, washed exhaustively with distilled water, hemozoin is insoluble (5). It has subsequently been proposed lyophilized, and further dried over P205. that hemozoin comprises an association of hematin with a j3-Hematin Synthesis. Sixty micromoles of hematin was protein, in which the protein could be partially degraded dissolved in 8 ml of 0.1 M NaOH, and the porphyrin was globin (7) or a parasite-derived polypeptide (8, 9). However, precipitated by the addition of 49 mmol of acetic acid a heme-protein type of structure for hemozoin is difficult to (benzoic, propionic, or succinic acid could substitute for reconcile with the finding that purified hemozoin is resistant acetic acid at this step). The suspension was heated overnight to nonspecific proteolysis and has an elemental composition at 70°C, and the precipitate was washed four times in distilled very similar to heme (10). This indicates that proteins asso- water. Unreacted hematin was removed by extracting the ciate nonspecifically with hemozoin during its isolation and precipitate twice for 3 hr in 0.1 M sodium bicarbonate buffer suggests that pure hemozoin is an insoluble derivative of at pH 9.1. The remaining insoluble material was recovered by hematin. centrifugation, washed four times in distilled water, lyoph- In this paper we describe the isolation and characterization ilized, and further dried over P205. This method routinely of hemozoin from trophozoites of the human malaria patho- converts 40-50% of the starting material into 83-hematin. In gen Plasmodiumfalciparum. The structure and properties of three experiments, 8-hematin was synthesized as above in The publication costs of this article were defrayed in part by page charge Abbreviation: EXAFS, extended x-ray absorption fine structure. payment. This article must therefore be hereby marked "advertisement" tTo whom reprint requests should be addressed. in accordance with 18 U.S.C. §1734 solely to indicate this fact. IDeceased September 24, 1990. 325 Downloaded by guest on September 30, 2021 326 Biochemistry: Slater et al. Proc. Natl. Acad. Sci. USA 88 (1991) the additional presence of 0.2 mCi (7.4 MBq) of [1,2- Table 1. Elemental compositions of hemozoin and )9-hematin 14C]acetic acid. The amount of heme in the final product was Percent by weight determined by the pyridine-hemochrome method (12), while radioactive acetic acid was detected by solubilizing the Hematin j3-hematin in 0.1 M NaOH, precipitating the heme with HCl, Element Hemozoin 1-Hematin (theory) and measuring the 14C in the supernatant by liquid scintilla- Carbon 63.7 64.7 64.5 tion counting. Hydrogen 5.6 5.0 5.3 Spectroscopy. Absorption spectra were recorded on a Hew- Nitrogen 7.5 8.7 8.8 lett-Packard 8450A UV/visible spectrophotometer. Mass Iron 7.5 8.3 8.8 spectra were obtained using a 252Cf time-of-flight fission- Chlorine <0.1 <0.3 - fragment mass spectrometer as described (13), with samples Oxygen* 15.6 13.3 12.6 of either hemozoin or hematin solubilized in 0.2 M NH40H. *As an accurate elemental analysis for oxygen cannot be performed To obtain IR spectra, KBr pellets were prepared from dried in the presence of significant amounts of iron, the oxygen values samples of each porphyrin, and spectra were acquired for 25 the cycles with a Fourier-transform IR spectrometer (Perkin- represent percent weight remaining. Elmer model 1800). X-ray powder diffraction patterns were strong base (see above), it is insoluble in weakly basic obtained using an x-ray generator (EnrafNonius FR 590) with bicarbonate or borate buffers (pH < 10.5; Fig. la). It is also a Cu Ka radiation source, operating at 50 kV and 40 mA for insoluble in aprotic solvents such as dimethyl sulfoxide (Fig. 30 min. ESR spectra were recorded at 10 K on a Bruker 200D lb) and pyridine. In contrast, hematin is readily solubilized in ESR spectrometer operating at a modulation frequency of100 both types of solvent (Fig. 1), either by solvation of its KHz, a microwave frequency of 9.52 GHz, and microwave carboxylic acid side chains (16) or by solvent coordination to power of 10 dB. the central ferric ion (17), respectively. The insolubility of Extended X-Ray Absorption Fine Structure (EXAFS). X-ray hemozoin in both basic and aprotic solvents therefore indi- absorption measurements were obtained from the Synchro- cates that modifications to the chemical environment of both tron Radiation Source (Daresbury, U.K.) operating at 2.0 the carboxylate side groups and the iron of its constituent GeV and a maximum current of 200 mA. The measurements hemes have occurred. were made at room temperature on station 8.1 operating in The chemical structure of intact hemozoin was investi- the fluorescence mode. Harmonic rejection was set at 70%o gated initially by Fourier-transform IR spectroscopy. The IR using a Si(III) monochromator. Three spectra were obtained spectrum obtained from hemozoin was significantly different from each sample investigated. The spectra were averaged, from that of either hematin (Fig. 2a) or hemin (unpublished k3-weighted, and Fourier-filtered from 1.0 to 4.6 A. Hemin data), although all of the spectra were clearly from closely was used as the model compound in the EXAFS analysis, related structures. The hemozoin spectrum was more sharply as its crystal structure is known (14). The EXCURV88 program resolved than that of hematin (indicating a reduction in was used to generate theoretical EXAFS spectra, which intermolecular hydrogen bonding), and contained additional were then compared to experimental results by using fit- major features at 1664 and 1211 cm-'.
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