Proc. Nati. Acad. Sci. USA Vol. 88, pp. 325-329, January 1991 Biochemistry An -carboxylate bond links the units of pigment (//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 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 of 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, (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 , 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 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-'. As strong absorbances index statistics (15). The Debye-Waller parameters (2o.2), between 1720 and 1650 cm-' in the IR spectra of protopor- representing the root-mean-square relative displacements of the atom pairs, were set at 0.017 A2 for N and at 0.015 A2 a 1.0 for all other atom pairs. 0.81 I RESULTS AND DISCUSSION 0.6 ? Hemozoin was purified from cultures of human erythrocytes infected with P. falciparum synchronized at the late tropho- 0.4- zoite stage. The UV/visible absorbance spectrum of an aqueous suspension of the purified material was similar to that of hemozoin in a crude parasite extract (unpublished 0.2 - data). An absorbance peak was present between 650 and 652 nm in each spectrum, which has been described previously as the characteristic feature of the absorbance spectrum of b 1.6 intact hemozoin (5, 10). As the solubility properties of the --a-- --a.- -,D..- -.*.- -0- purified material (see below) are also identical to those -.0- -0--o described for hemozoin in a crude parasite extract (5), we are 1.2 confident that the purification process did not significantly alter the structure and properties of hemozoin. N The elemental composition of purified hemozoin was very v 0.8 similar to that of hematin (Table 1), supporting results obtained with hemozoin isolated from the rodent malaria 0.4 I parasite Plasmodium berghei (10). The UV/visible absor- 0 0 bance spectrum of hemozoin solubilized in 0.1 M NaOH was identical to that of hematin (unpublished data). When the 0.0 I I solubilized material was analyzed by mass spectrometry, its 0 12 24 36 48 60 molecular ion was at m/z 616.3, again identical to the Time, min molecular ion obtained with base-solubilized hematin. This structure of hemozoin is FIG. 1. Comparison ofthe solubility ofhemozoin (e) and hematin all indicates that the chemical (o) in 0.1 M bicarbonate at pH 10.2 (a) and in dimethyl sulfoxide (b). closely related to that of heme and does not include a In each experiment, 30 ,umol of an aqueous suspension of hematin or proteinaceous component. hemozoin was added, and the absorbance of solubilized heme was To investigate the physical properties of the purified pig- measured at 387 or 402 nm in bicarbonate or dimethyl sulfoxide, ment, its solubility in aqueous basic and aprotic solvents was respectively. Average absorbances from three experiments are pre- studied. Although hemozoin is hydrolyzed to hematin in sented in each case. Downloaded by guest on September 30, 2021 Biochemistry: Slater et al. Proc. Natl. Acad. Sci. USA 88 (1991) 327 a

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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 from (23). As a transition from high to low spin state involves an their carboxylate groups (18, 19), a comparison ofthe spectra increase in the electric field strength surrounding the ferric in this region indicates a substantial difference in the chem- ion (24), these spectra are consistent with the axial hydroxyl istry of the carboxylates of hemozoin compared with hema- or chloride anion in hematin or hemin being replaced by a tin. stronger r-bonding ligand such as a carboxylate in hemozoin. A possible structure for hemozoin that would account for The local environment of iron in hemozoin was further both its observed insolubility and its IR spectrum is a direct investigated using EXAFS, a technique that allows both the coordination between a carboxylate ofone heme and the iron type and the distance of each shell of atoms around the of another. An extensive series of iron(III) carboxylate absorbing metal to be identified (25). The EXAFS spectra compounds have been described, in which either one (uni- obtained from hemin and hemozoin were different (Fig. 3a), dentate) or both (symmetrical chelate) of the carboxylate indicating that a difference in the atoms surrounding iron are coordinated to the metal. When the coordination exists between the two compounds. Interatomic distances is unidentate, one of the C-0 bonds is predicted to have from a published crystal structure (14) were used to produce double-bond character and should therefore give rise to a a theoretical model EXAFS of hemin (Fig. 3b). Fe-N and C=O stretching absorption between 1700 and 1600 cm-' in Fe-Cl bond distances were each varied over a short range, the IR region. For example, acetato[deuteroporphyrin IX and their optima were found at 2.047 A and 2.212 A, respec- dimethyl ester]iron(III) has an acetate C=O stretching fre- tively (Fig. 4a). This compares very closely to the crystal quency at 1660 cm-' (20), typical of a ferric ion with a structure estimates of 2.06 + 0.01 and 2.218 A. unidentate acetate coordination. The IR spectra ofhemozoin The hemin model gave a poor fit to the experimental contains an intense absorbance at 1664 cm-' that is absent EXAFS of hemozoin (fit index = 5.20). A large improvement from the spectra of either hemin or hematin. This strongly in the model was obtained when the chlorine atom was suggests the presence of a unidentate carboxylate coordina- replaced with a single oxygen atom, at an optimal distance of tion onto iron in this pigment. The other strong distinguishing 1.92 A (fit index = 1.36; Fig. 4b). This model worsened when IR absorbance in hemozoin (at 1211 cm-') can also be two oxygen atoms were present (fit index = 3.08), although assigned to an axial carboxylate ligand, as C-0 stretching it improved when a single carbon atom was placed at 2.17 A frequencies from 0-methyl groups linked to various metal- (fit index = 1.27; Fig. 4b). A further improvement occurred loporphyrins are known to lie between 1270 and 1080 cm-' when a second oxygen shell, containing one atom, was (18, 21). In addition, since axial Fe-C(O) coordinations introduced at 3.7 A (fit index = 1.23; Fig. 4c). A comparison produce very strong C-0 stretching frequencies around ofthe EXAFS produced by the final model with that obtained 1900 cm-1 (22), we can discount a direct iron-carbon linkage from hemozoin is shown in Fig. 3c. As our IR analysis makes from the structure of hemozoin. a direct iron-carbon linkage unlikely, these oxygen and ESR spectroscopy was used to characterize the charge and carbon atoms probably belong to a single carboxylate func- spin state of iron in hemozoin. At 10 K, hemozoin gave an tional group in which only the first-shell oxygen is directly ESR spectrum indicative of low-spin ferric iron (dominant coordinated to the iron. EXAFS spectra were also obtained signals at g = 3.80 and 1.95), while as reported previously, the from hematin, and a similar modeling analysis was under- iron in both hematin and hemin was in a high-spin ferric state taken (unpublished data). In hematin, a single oxygen atom Downloaded by guest on September 30, 2021 328 Biochemistry: Slater et al. Proc. Natl. Acad. Sci. USA 88 (1991)

a 7 6-

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:t- FIG. 3. EXAFS comparisons. (a) Hemin (-) and hemozoin LL --- -). (b) Hemin (-) and the best-fit hemin model (--- -; fit index = 1.45). (c) Hemozoin (-) and the best-fit hemozoin model (- - -; fit index = 1.23). For the model, the approximate position ofeach shell ofcarbon atoms in the porphyrin ring was obtained (14) and adjusted slightly to 3.000 A (four atoms), 3.110 A (four atoms), 3.395 A (four atoms), and 4.34 A (eight atoms) from the iron. 3.4 3.5 3.6 3.7 3.8 3.9 was detected 1.99 A from the iron (belonging to the hydroxyl Distance, A axial ligand), but as expected, further single shells of carbon or oxygen atoms could not be located. FIG. 4. Variation in the fit-index statistic for theoretical EXAFS Previous cystallographic studies of iron-carboxylate com- compared with experimental hemin EXAFS (a) and experimental hemozoin EXAFS (b and c). In a, Fe-N (r, with Fe-Cl distances = pounds, which all were concerned with non-heme symmetric 2.21 A) or Fe-Cl (*, with Fe-N distances = 2.04 A) distances were chelations, have found Fe-O bond lengths between 2.00 and varied. In b, Fe-ist 0 (c) and then Fe-ist C (*, with Fe-ist 0 = 1.92 2.05 A (26-28). In hemozoin, in which the iron-carboxylate A) shell distances were sequentially varied. In c, Fe-2nd 0 (o, with coordination is unidentate, we estimate the shortest Fe-O Fe-ist 0 = 1.92 A and Fe-ist C = 2.17 A) shell distance was varied. distance to be 1.92 A. Further, if we assume that (i) the angle Positions of the other shells of carbon atoms in the model are given OCO in a typical carboxylate is 1200, (ii) the C-O and C=O in the legend to Fig. 3. bond lengths in a typical carboxylate are 1.43 and 1.23 A, respectively, and (iii) the Fe-ist 0 and Fe-ist C shell bond powder diffraction pattern obtained from 83-hematin was lengths are as measured, then the Fe-2nd 0 shell bond length indistinguishable from that of hemozoin (Fig. 5), providing is calculated at 3.4 A. This is close to our experimental value further evidence that these compounds have an identical of 3.7 A, consistent with the structure proposed. structure and showing that both possess an ordered micro- Fitch and Kanjananggulpan (10) observed that the solubil- crystalline state. As the synthesis of f3-hematin requires ity properties ofhemozoin are similar to those of a derivative acetate concentrations in excess of 3 M, buffering below pH of hematin (called p-hematin) first described by Hamsik (29) 4.5, and an elevated temperature, a structure of this type over 50 years ago. To examine the possibility that the could not have formed under the conditions used to purify chemical structure of f3-hematin is identical with that of hemozoin and therefore corresponds to hemozoin in situ. hemozoin, a base-insoluble pigment was synthesized by The axial carboxylate liganded to each ferric ion in hemo- heating hematin in dilute acetic acid. The product had an zoin could either come from one of the two propionate side elemental composition (Table 1), IR spectrum (Fig. 2b) and chains of a second heme or belong to an extraneous carbox- EXAFS spectrum (fit index = 1.33 when compared to the ylate. When 83-hematin was synthesized in the presence of hemozoin model) very similar to that of hemozoin. The x-ray [14C]acetic acid, <0.05 mol of acetic acid was recovered per Downloaded by guest on September 30, 2021 Biochemistry: Slater et al. Proc. Natl. Acad. Sci. USA 88 (1991) 329 Hemozoin f3-Hematin

......

u.. *....; o . g.. ., ...... ,;#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). 6. Brown, W. H. (1911) J. Exp. Med. 13, 290-299. The failure to stochiometrically incorporate acetate into the 7. Sherman, I. W., Ting, I. P. & Ruble, J. A. (1968) J. Protozool. synthetic pigment shows that the carboxylate bonded to each 15, 158-164. 8. Yamada, K. A. & Sherman, I. W. (1979) Exp. Parasitol. 48, ferric ion of both hemozoin and 83-hematin must come from 61-74. a propionate side chain of another heme. This is consistent 9. Ashong, J. O., Blench, I. P. & Warhurst, D. C. (1989) Trans. with the elemental analysis and solubility properties of both R. Soc. Trop. Med. Hyg. 83, 167-172. compounds. An iron-carboxylate linkage between adjacent 10. Fitch, C. D. & Kanjananggulpan, P. (1987) J. Biol. Chem. 262, hemes allows two or more monomers to polymerize into 15552-15555. hemozoin, although the exact number ofhemes in each chain 11. Trager, W. & Jensen, J. B. (1976) Science 193, 673-675. in unknown. 12. Fuhrhop, J.-H. & Smith, K. M. (1975) in and Recently we have shown that Metalloporphyrins, ed. Smith, K. M. (Elsevier, Amsterdam), hemoglobin proteolysis in the pp. 757-889. food vacuole of a malaria trophozoite is a highly ordered 13. Chait, B. T. & Field, F. H. (1986) Biochem. Biophys. Res. process (30), and as free heme has not been detected in the Commun. 134, 420-426. parasite, it must be coupled to an efficient mechanism of 14. Koenig, D. F. (1965) Acta Crystallogr. 18, 663-673. forming hemozoin. It is probable that hemozoin biosynthesis 15. Gurman, S. J., Binstead, N. & Ross, I. (1984) J. Phys. C. Solid is -catalyzed, although an enzyme reaction mecha- State Phys. 17, 143-151. nism by which hemes could be polymerized through an 16. Falk, J. E. (1964) Porphyrins and Metalloporphyrins (Elsevier, iron-carboxylate bond has not been described. A full eluci- Amsterdam), p. 115. dation of this pathway will significantly advance our under- 17. Brown, S. B. & Lantzke, I. R. (1969) Biochem. J. 115, 279- of malaria and a new 285. standing biochemistry may provide 18. Burger, H. (1975) in Porphyrins and Metalloporphyrins, ed. target for selective chemotherapy against these important Smith, K. M. (Elsevier, Amsterdam), pp. 525-535. human pathogens. 19. Alben, J. 0. (1978) in The Porphyrins, ed. Dolphin, D. (Aca- demic, New York), Vol. 3, pp. 323-345. We dedicate this paper to the memory ofGraeme Henderson, who 20. Sadasivan, N., Eberspaecher, H. I., Fuchsman, W. H. & died on September 24, 1990, from injuries sustained in a hit-and-run Caughey, W. S. (1969) Biochemistry 8, 534-541. traffic accident. We thank Trevor Slater for helpful discussion. We 21. Buchler, J. W., Puppe, L., Rohbock, K. & Schneehage, H. H. are most grateful to Dr. B. Chait and T. Chowdary for performing the (1973) Chem. Ber. 106, 2710-2732. mass spectroscopy; Dr. R. Cammack for assistance with the ESR; 22. Cotton, F. A. & Wilkinson, G. (1980) in Advanced Inorganic and Drs. S. Hasnain, G. P. Diakum, and R. Bilsbarrow for help with Chemistry (Wiley, New York), p. 1072. the EXAFS. This work was supported by Grant RF 88062 from the 23. Schoffa, G. (1964) Nature (London) 203, 640-641. Rockefeller Foundation. 24. Subramanian, J. (1975) in Porphyrins and Metalloporphyrins, ed. Smith, K. M. (Elsevier, Amsterdam), pp. 555-589. 1. Scheibel, L. W. & Sherman, I. W. (1988) in Malaria: Principles 25. Powers, L. (1982) Biochim. Biophys. Acta 683, 1-38. and Practice of Malariology, eds. Wernsdorfer, W. H. & 26. Armstrong, W. H., Spool, A., Papaefthymiou, G. C., Frankel, McGregor, I. (Churchill Livingstone, Edinburgh, U.K.), pp. R. B. & Lippard, S. J. (1984) J. Am. Chem. Soc. 106, 3653- 219-252. 3667. 2. Rudzinska, M. A., Trager, W. & Bray, R. S. (1965) J. Proto- 27. Armstrong, W. H. & Lippard, S. J. (1985) J. Am. Chem. Soc. zool. 12, 563-576. 107, 3730-3731. 3. Tappel, A. L. (1953) Arch. Biochem. Biophys. 44, 378-395. 28. Wieghardt, K., Pohl, K. & Gebert, W. (1983) Angew. Chem. 4. Eckman, J. R., Modler, S., Eaton, J. W., Berger, E. & Engel, Int. Ed. Engl. 22, 727. R. R. (1977) J. Lab. Clin. Med. 90, 767-770. 29. Hamsik, A. (1936) Z. Physiol. Chem. 190, 199-215. 5. Deegan, T. & Maegraith, B. G. (1956) Ann. Trop. Med. Para- 30. Goldberg, D. E., Slater, A. F. G., Cerami, A. & Henderson, sitol. 50, 194-211. G. B. (1990) Proc. Natl. Acad. Sci. USA 87, 2931-2935. Downloaded by guest on September 30, 2021