Proc. Nati. Acad. Sci. USAVol. 88, pp. 325-329, January 1991Biochemistry

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 Biologyand Biochemistry, Brunel University, Uxbridge, United Kingdom

Communicated by Maclyn McCarty, October 15, 1990 (received for review August 17, 1990)

ABSTRACT The intraerythrocytic malaria parasite useshemoglobin as a major nutrient source. Digestion of hemoglo-bin releases heme, which the parasite converts into an insolublemicrocrystalline material called hemozoin or malaria pigment.We have purified hemozoin from the human malaria organismPlasmodium falkiparum and have used infrared spectroscopy,x-ray absorption spectroscopy, and chemical synthesis to de-termine its structure. The molecule consists of an unusualpolymer of hemes linked between the central ferric ion of oneheme and a carboxylate side-group oxygen of another. Thehemes are sequestered via this linkage into an insoluble prod-uct, providing a unique way for the malaria parasite to avoidthe toxicity associated with soluble heme.

The dark brown discoloration of the liver, spleen, and brainobserved in patients suffering a severe malaria infection wasfirst reported by Lancisi in 1717 (1), long before the parasiteitself was described. The crystalline substance causing thisdiscoloration (called malaria pigment or hemozoin) is formedwithin the food vacuoles of intraerythrocytic malaria para-sites as a product of the catabolism of hemoglobin (2).Proteolysis of hemoglobin releases heme, which when solu-ble is highly toxic to biological membranes (3). As malariaparasites lack heme oxygenase, they are unable to cleaveheme into an open-chain tetrapyrrole (4), and it is notexcreted from the cell. Instead heme is detoxified by con-version into hemozoin, a process unique to the malariaorganism. Hemozoin is released along with the merozoiteswhen the infected erythrocytes burst and is scavenged bymacrophages. This pigment is insoluble under physiologicconditions and remains undegraded within tissue macro-phages of the host for an extended period of time (5).The chemical nature of hemozoin has been the subject of

study and speculation since Brown (6) first remarked on thesimilarity between hemozoin and hematin (ferriprotoporphy-rin IX hydroxide). However, hemozoin is not identical tohematin, as hematin dissolves rapidly in solvents in whichhemozoin is insoluble (5). It has subsequently been proposedthat hemozoin comprises an association of hematin with aprotein, in which the protein could be partially degradedglobin (7) or a parasite-derived polypeptide (8, 9). However,a heme-protein type of structure for hemozoin is difficult toreconcile with the finding that purified hemozoin is resistantto nonspecific proteolysis and has an elemental compositionvery similar to heme (10). This indicates that proteins asso-ciate nonspecifically with hemozoin during its isolation andsuggests that pure hemozoin is an insoluble derivative ofhematin.

In this paper we describe the isolation and characterizationof hemozoin from trophozoites of the human malaria patho-gen Plasmodiumfalciparum. The structure and properties of

the purified pigment are shown to be identical to those ofhemozoin in situ. Using chemical synthesis and IR, ESR, andx-ray absorption spectroscopy we demonstrate that hemo-zoin consists of heme moieties linked by a bond between theferric ion of one heme and a carboxylate side-group oxygenof another. This linkage allows the heme units released byhemoglobin breakdown to aggregate into an ordered insolu-ble product and represents a novel way for the parasite toavoid the toxicity associated with soluble hematin.

MATERIALS AND METHODSMaterials. Hematin, hemin (ferriprotoporphyrin IX chlo-

ride), dimethyl sulfoxide, pyridine, and KBr were obtainedfrom Aldrich. Sodium [1,2-14C]acetate was supplied by NEN.All other chemicals and reagents were purchased fromSigma. Elemental analyses were performed by SchwarzkopfMicroanalytical Laboratory (Woodside, NY).

Parasite Culture. P. falciparum clone HB-3 was cultured inA+ human erythrocytes by the method ofTrager and Jensen(11). Synchrony was maintained by sorbitol treatment, andlate trophozoite-stage parasites were harvested by saponinlysis.Hemozoin Purification. A crude extract of hemozoin was

prepared as described (8). In all subsequent steps, hemozoinwas suspended into buffer by brief sonication and thenremoved from solubilized contaminants by centrifugation at25,000 x g for 30 min at 4°C. To solubilize any contaminatingmembranes, crude hemozoin was extracted twice for 3 hr atroom temperature in buffer (50 mM Tris-HCI, pH 7.4) con-taining 2% sodium dodecyl sulfate. The hemozoin pellet waswashed three times in buffer, and residual proteins wereremoved by an overnight digestion in buffer containingproteinase E at 1 mg/ml. Insoluble material was recoveredand washed as above, before being extracted in aqueous 6 Murea for 3 hr at 4°C. The purified hemozoin was pelleted bycentrifugation, washed exhaustively with distilled water,lyophilized, and further dried over P205.

j3-Hematin Synthesis. Sixty micromoles of hematin wasdissolved in 8 ml of 0.1 M NaOH, and the porphyrin wasprecipitated by the addition of 49 mmol of acetic acid(benzoic, propionic, or succinic acid could substitute foracetic acid at this step). The suspension was heated overnightat 70°C, and the precipitate was washed four times in distilledwater. Unreacted hematin was removed by extracting theprecipitate twice for 3 hr in 0.1 M sodium bicarbonate bufferat pH 9.1. The remaining insoluble material was recovered bycentrifugation, washed four times in distilled water, lyoph-ilized, and further dried over P205. This method routinelyconverts 40-50% of the starting material into 83-hematin. Inthree experiments, 8-hematin was synthesized as above in

Abbreviation: EXAFS, extended x-ray absorption fine structure.tTo whom reprint requests should be addressed.IDeceased September 24, 1990.

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the additional presence of 0.2 mCi (7.4 MBq) of [1,2-14C]acetic acid. The amount of heme in the final product wasdetermined by the pyridine-hemochrome method (12), whileradioactive acetic acid was detected by solubilizing thej3-hematin in 0.1 M NaOH, precipitating the heme with HCl,and measuring the 14C in the supernatant by liquid scintilla-tion counting.

Spectroscopy. Absorption spectra were recorded on a Hew-lett-Packard 8450A UV/visible spectrophotometer. Massspectra were obtained using a 252Cf time-of-flight fission-fragment mass spectrometer as described (13), with samplesof either hemozoin or hematin solubilized in 0.2 M NH40H.To obtain IR spectra, KBr pellets were prepared from driedsamples of each porphyrin, and spectra were acquired for 25cycles with a Fourier-transform IR spectrometer (Perkin-Elmer model 1800). X-ray powder diffraction patterns wereobtained using an x-ray generator (EnrafNonius FR 590) witha Cu Ka radiation source, operating at 50 kV and 40 mA for30 min. ESR spectra were recorded at 10 K on a Bruker 200DESR spectrometer operating at a modulation frequency of 100KHz, a microwave frequency of 9.52 GHz, and microwavepower of 10 dB.Extended X-Ray Absorption Fine Structure (EXAFS). X-ray

absorption measurements were obtained from the Synchro-tron Radiation Source (Daresbury, U.K.) operating at 2.0GeV and a maximum current of 200 mA. The measurementswere made at room temperature on station 8.1 operating inthe fluorescence mode. Harmonic rejection was set at 70%ousing a Si(III) monochromator. Three spectra were obtainedfrom each sample investigated. The spectra were averaged,k3-weighted, and Fourier-filtered from 1.0 to 4.6 A. Heminwas used as the model compound in the EXAFS analysis,as its crystal structure is known (14). The EXCURV88 programwas used to generate theoretical EXAFS spectra, whichwere then compared to experimental results by using fit-index statistics (15). The Debye-Waller parameters (2o.2),representing the root-mean-square relative displacements ofthe atom pairs, were set at 0.017 A2 for N and at 0.015 A2for all other atom pairs.

RESULTS AND DISCUSSIONHemozoin was purified from cultures ofhuman erythrocytesinfected with P. falciparum synchronized at the late tropho-zoite stage. The UV/visible absorbance spectrum of anaqueous suspension of the purified material was similar tothat of hemozoin in a crude parasite extract (unpublisheddata). An absorbance peak was present between 650 and 652nm in each spectrum, which has been described previously asthe characteristic feature of the absorbance spectrum ofintact hemozoin (5, 10). As the solubility properties of thepurified material (see below) are also identical to thosedescribed for hemozoin in a crude parasite extract (5), we areconfident that the purification process did not significantlyalter the structure and properties of hemozoin.The elemental composition of purified hemozoin was very

similar to that of hematin (Table 1), supporting resultsobtained with hemozoin isolated from the rodent malariaparasite Plasmodium berghei (10). The UV/visible absor-bance spectrum of hemozoin solubilized in 0.1 M NaOH wasidentical to that of hematin (unpublished data). When thesolubilized material was analyzed by mass spectrometry, itsmolecular ion was at m/z 616.3, again identical to themolecular ion obtained with base-solubilized hematin. Thisall indicates that the chemical structure of hemozoin isclosely related to that of heme and does not include aproteinaceous component.To investigate the physical properties of the purified pig-

ment, its solubility in aqueous basic and aprotic solvents wasstudied. Although hemozoin is hydrolyzed to hematin in

Table 1. Elemental compositions of hemozoin and )9-hematinPercent by weight

HematinElement Hemozoin 1-Hematin (theory)

Carbon 63.7 64.7 64.5Hydrogen 5.6 5.0 5.3Nitrogen 7.5 8.7 8.8Iron 7.5 8.3 8.8Chlorine <0.1 <0.3 -

Oxygen* 15.6 13.3 12.6

*As an accurate elemental analysis for oxygen cannot be performedin the presence of significant amounts of iron, the oxygen valuesrepresent the percent weight remaining.

strong base (see above), it is insoluble in weakly basicbicarbonate or borate buffers (pH < 10.5; Fig. la). It is alsoinsoluble in aprotic solvents such as dimethyl sulfoxide (Fig.lb) and pyridine. In contrast, hematin is readily solubilized inboth types of solvent (Fig. 1), either by solvation of itscarboxylic acid side chains (16) or by solvent coordination tothe central ferric ion (17), respectively. The insolubility ofhemozoin in both basic and aprotic solvents therefore indi-cates that modifications to the chemical environment of boththe carboxylate side groups and the iron of its constituenthemes have occurred.The chemical structure of intact hemozoin was investi-

gated initially by Fourier-transform IR spectroscopy. The IRspectrum obtained from hemozoin was significantly differentfrom that of either hematin (Fig. 2a) or hemin (unpublisheddata), although all of the spectra were clearly from closelyrelated structures. The hemozoin spectrum was more sharplyresolved than that of hematin (indicating a reduction inintermolecular hydrogen bonding), and contained additionalmajor features at 1664 and 1211 cm-'. As strong absorbancesbetween 1720 and 1650 cm-' in the IR spectra of protopor-

a 1.0

0.81

0.6 ?

0.4-

0.2 -

b 1.6

1.2

N

v 0.8

0.4

0.00 12 24 36 48

Time, min60

FIG. 1. Comparison ofthe solubility ofhemozoin (e) and hematin(o) in 0.1 M bicarbonate at pH 10.2 (a) and in dimethyl sulfoxide (b).In each experiment, 30 ,umol of an aqueous suspension of hematin orhemozoin was added, and the absorbance of solubilized heme wasmeasured at 387 or 402 nm in bicarbonate or dimethyl sulfoxide,respectively. Average absorbances from three experiments are pre-sented in each case.

--a-- --a.- -,D..- -.*.--0- -.0-

-0--o

I0 0

I I

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a

O

0-a)

a)c:

b

O

a)._

cr

cm-1

FIG. 2. Comparisons of Fourier-transform IR spectra. (a) Hemozoin (-) and hematin (--- -). (b) Hemozoin (-) and ,8-hematin (... ). T,transmittance.

phyrins can be assigned to C=-O stretching vibrations fromtheir carboxylate groups (18, 19), a comparison of the spectrain this region indicates a substantial difference in the chem-istry of the carboxylates of hemozoin compared with hema-tin.A possible structure for hemozoin that would account for

both its observed insolubility and its IR spectrum is a directcoordination between a carboxylate ofone heme and the ironof another. An extensive series of iron(III) carboxylatecompounds have been described, in which either one (uni-dentate) or both (symmetrical chelate) of the carboxylateoxygens are coordinated to the metal. When the coordinationis unidentate, one of the C-0 bonds is predicted to havedouble-bond character and should therefore give rise to aC=O stretching absorption between 1700 and 1600 cm-' inthe IR region. For example, acetato[deuteroporphyrin IXdimethyl ester]iron(III) has an acetate C=O stretching fre-quency at 1660 cm-' (20), typical of a ferric ion with aunidentate acetate coordination. The IR spectra of hemozoincontains an intense absorbance at 1664 cm-' that is absentfrom the spectra of either hemin or hematin. This stronglysuggests the presence of a unidentate carboxylate coordina-tion onto iron in this pigment. The other strong distinguishingIR absorbance in hemozoin (at 1211 cm-') can also beassigned to an axial carboxylate ligand, as C-0 stretchingfrequencies from 0-methyl groups linked to various metal-loporphyrins are known to lie between 1270 and 1080 cm-'(18, 21). In addition, since axial Fe-C(O) coordinationsproduce very strong C-0 stretching frequencies around1900 cm-1 (22), we can discount a direct iron-carbon linkagefrom the structure of hemozoin.ESR spectroscopy was used to characterize the charge and

spin state of iron in hemozoin. At 10 K, hemozoin gave anESR spectrum indicative of low-spin ferric iron (dominantsignals at g = 3.80 and 1.95), while as reported previously, theiron in both hematin and hemin was in a high-spin ferric state

(23). As a transition from high to low spin state involves anincrease in the electric field strength surrounding the ferricion (24), these spectra are consistent with the axial hydroxylor chloride anion in hematin or hemin being replaced by astronger r-bonding ligand such as a carboxylate in hemozoin.The local environment of iron in hemozoin was further

investigated using EXAFS, a technique that allows both thetype and the distance of each shell of atoms around theabsorbing metal to be identified (25). The EXAFS spectraobtained from hemin and hemozoin were different (Fig. 3a),indicating that a difference in the atoms surrounding ironexists between the two compounds. Interatomic distancesfrom a published crystal structure (14) were used to producea theoretical model EXAFS of hemin (Fig. 3b). Fe-N andFe-Cl bond distances were each varied over a short range,and their optima were found at 2.047 A and 2.212 A, respec-tively (Fig. 4a). This compares very closely to the crystalstructure estimates of 2.06 + 0.01 and 2.218 A.The hemin model gave a poor fit to the experimental

EXAFS of hemozoin (fit index = 5.20). A large improvementin the model was obtained when the chlorine atom wasreplaced with a single oxygen atom, at an optimal distance of1.92 A (fit index = 1.36; Fig. 4b). This model worsened whentwo oxygen atoms were present (fit index = 3.08), althoughit improved when a single carbon atom was placed at 2.17 A(fit index = 1.27; Fig. 4b). A further improvement occurredwhen a second oxygen shell, containing one atom, wasintroduced at 3.7 A (fit index = 1.23; Fig. 4c). A comparisonof the EXAFS produced by the final model with that obtainedfrom hemozoin is shown in Fig. 3c. As our IR analysis makesa direct iron-carbon linkage unlikely, these oxygen andcarbon atoms probably belong to a single carboxylate func-tional group in which only the first-shell oxygen is directlycoordinated to the iron. EXAFS spectra were also obtainedfrom hematin, and a similar modeling analysis was under-taken (unpublished data). In hematin, a single oxygen atom

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a 7

6-

5-x4)VD 4 -

.t_

IL 3-

2-

1

b 2.6

2.4 -

2.2 -

xa)

-o._

FIG. 3. EXAFS comparisons. (a) Hemin (-) and hemozoin--- -). (b) Hemin (-) and the best-fit hemin model (--- -; fit index =1.45). (c) Hemozoin (-) and the best-fit hemozoin model (- - -; fitindex = 1.23). For the model, the approximate position of each shellof carbon atoms in the porphyrin ring was obtained (14) and adjustedslightly to 3.000 A (four atoms), 3.110 A (four atoms), 3.395 A (fouratoms), and 4.34 A (eight atoms) from the iron.

was detected 1.99 A from the iron (belonging to the hydroxylaxial ligand), but as expected, further single shells of carbonor oxygen atoms could not be located.

Previous cystallographic studies of iron-carboxylate com-pounds, which all were concerned with non-heme symmetricchelations, have found Fe-O bond lengths between 2.00 and2.05 A (26-28). In hemozoin, in which the iron-carboxylatecoordination is unidentate, we estimate the shortest Fe-Odistance to be 1.92 A. Further, ifwe assume that (i) the angleOCO in a typical carboxylate is 1200, (ii) the C-O and C=Obond lengths in a typical carboxylate are 1.43 and 1.23 A,respectively, and (iii) the Fe-ist 0 and Fe-ist C shell bondlengths are as measured, then the Fe-2nd 0 shell bond lengthis calculated at 3.4 A. This is close to our experimental valueof 3.7 A, consistent with the structure proposed.

Fitch and Kanjananggulpan (10) observed that the solubil-ity properties of hemozoin are similar to those of a derivativeof hematin (called p-hematin) first described by Hamsik (29)over 50 years ago. To examine the possibility that thechemical structure of f3-hematin is identical with that ofhemozoin, a base-insoluble pigment was synthesized byheating hematin in dilute acetic acid. The product had anelemental composition (Table 1), IR spectrum (Fig. 2b) andEXAFS spectrum (fit index = 1.33 when compared to thehemozoin model) very similar to that of hemozoin. The x-ray

2.0

1.8

1.6

1.4

1.2

c

xa)

:t-LL

I I I I.

1.9 2.0 2.1 2.2 2.3

3.4 3.5 3.6 3.7Distance, A

2.4

3.8 3.9

FIG. 4. Variation in the fit-index statistic for theoretical EXAFScompared with experimental hemin EXAFS (a) and experimentalhemozoin EXAFS (b and c). In a, Fe-N (r, with Fe-Cl distances =2.21 A) or Fe-Cl (*, with Fe-N distances = 2.04 A) distances werevaried. In b, Fe-ist 0 (c) and then Fe-ist C (*, with Fe-ist0 = 1.92A) shell distances were sequentially varied. In c, Fe-2nd 0 (o, withFe-ist 0 = 1.92 A and Fe-ist C = 2.17 A) shell distance was varied.Positions of the other shells of carbon atoms in the model are givenin the legend to Fig. 3.

powder diffraction pattern obtained from 83-hematin wasindistinguishable from that of hemozoin (Fig. 5), providingfurther evidence that these compounds have an identicalstructure and showing that both possess an ordered micro-crystalline state. As the synthesis of f3-hematin requiresacetate concentrations in excess of 3 M, buffering below pH4.5, and an elevated temperature, a structure of this typecould not have formed under the conditions used to purifyhemozoin and therefore corresponds to hemozoin in situ.The axial carboxylate liganded to each ferric ion in hemo-

zoin could either come from one of the two propionate sidechains of a second heme or belong to an extraneous carbox-ylate. When 83-hematin was synthesized in the presence of[14C]acetic acid, <0.05 mol of acetic acid was recovered per

VP

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Hemozoin f3-Hematin

.. ..

.... ....... . ..u..

.; g .,

* o.......... .. . . . #.,;#F ,.

FIG. 5. X-ray powder diffraction patterns of hemozoin (Left) and 9-hematin (Right).

mol ofheme (three experiments, mean = 0.043, SD = 0.015).The failure to stochiometrically incorporate acetate into thesynthetic pigment shows that the carboxylate bonded to eachferric ion of both hemozoin and 83-hematin must come froma propionate side chain of another heme. This is consistentwith the elemental analysis and solubility properties of bothcompounds. An iron-carboxylate linkage between adjacenthemes allows two or more monomers to polymerize intohemozoin, although the exact number of hemes in each chainin unknown.Recently we have shown that hemoglobin proteolysis in the

food vacuole of a malaria trophozoite is a highly orderedprocess (30), and as free heme has not been detected in theparasite, it must be coupled to an efficient mechanism offorming hemozoin. It is probable that hemozoin biosynthesisis enzyme-catalyzed, although an enzyme reaction mecha-nism by which hemes could be polymerized through aniron-carboxylate bond has not been described. A full eluci-dation of this pathway will significantly advance our under-standing of malaria biochemistry and may provide a newtarget for selective chemotherapy against these importanthuman pathogens.

We dedicate this paper to the memory of Graeme Henderson, whodied on September 24, 1990, from injuries sustained in a hit-and-runtraffic accident. We thank Trevor Slater for helpful discussion. Weare most grateful to Dr. B. Chait and T. Chowdary for performing themass spectroscopy; Dr. R. Cammack for assistance with the ESR;and Drs. S. Hasnain, G. P. Diakum, and R. Bilsbarrow for help withthe EXAFS. This work was supported by Grant RF 88062 from theRockefeller Foundation.

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