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A simple magnetic method for the purification of malarial pigment
Molecular and Biochemical Parasitology, 12 (1984) 307-312 Elsevier
307
MBP 00460
A SIMPLE MAGNETIC METHOD FOR THE PURIFICATION OF MALARIAL PIGMENT
ALAN H. FAIRLAMB 1'*, FRANK PAUL 2 and DAVID C. WARHURST ~
~Department of Medical Protozoology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, and 2Tenovus Research Institute, Tremona Road, Southampton S09 4XY, England.
(Received 11 January 1984; accepted 22 March 1984)
Using a simple magnetic separation apparatus, Plasmodium berghei malarial pigment has been purified 200-fold in a single step. The technique exploits the paramagnetic properties of malarial pigment (hemozoin) and provides a simple and rapid method for the isolation of this material with high purification and
yield. Key words: Malarial pigment; Hemozoin; Plasmodium berghei; Purification; Magnetic technique
INTRODUCTION
Erythrocyte hemoglobin provides a major source of amino acids for the growth of the intracellular form of the malarial parasite. The end product of hemoglobin digestion within the food vacuole is malarial pigment or hemozoin, which can be readily seen as golden brown-black deposits in the trophozoite and schizont stages of the parasite (reviewed in [ 1,2]). After solubilization with alkali, the main constituent of malarial pigment can be isolated as hematin [3-5], but in the insoluble native state, the iron porphyrin may be associated with protein [5] to form regular crystalline arrays [6]. Malarial pigment may represent the parasites' means of sequestering free heme in an inert, non-toxic form [1]. The mechanism by which heme is complexed as malarial pigment is of interest from a chemotherapeutic standpoint, since unsequestered heine may represent the high-affinity binding site for the antimalarial drug chloroquine [7]. Moreover, the drug's antimalarial action may reside in the fact that chloroquine-ferriprotoporphyrin IX complexes lyse the malarial parasite [8,9]. High-spin hematin and consequently malarial pigment are paramagnetic, in contrast to low-spin oxyhemoglobin of the uninfected erythrocyte, which is diamag-
Abbreviation: STE buffer, 250 mM sucrose, 1 mM EDTA, 25 mM Tris-HCl, pH 7.4. *Present address: Laboratory of Medical Biochemistry, The Rockefeller University, 1230 York Avenue, New York, NY 10021, U.S.A. 0166-6851/84/$03.00 © 1984 Elsevier Science Publishers B.V.
308 netic. This difference in the magnetic properties of infected and uninfected erythrocytes has been exploited to selectively concentrate red cells infected with late-stage parasites from whole blood [10,11]. In this report, we show that this technique can be applied to the isolation of malarial pigment itself. MATERIALS AND METHODS
Preparation of parasite homogenate. Mice infected with Plasmodium berghei (N strain) 4 days previously were bled by cardiac puncture under ether anesthesia and the infected blood collected in heparinized Krebs' Ringer saline. After washing twice with Krebs' Ringer solution by centrifugation (500 X g, 10 min) the red blood cells (22% parasitemia) were passed through a CF- 11 cellulose column to remove contaminating white blood cells and platelets [ 12]. The infected blood (4 ml packed cells) was washed once in 250 mM sucrose, 1 mM E D T A , 25 mM Tris-HC1, pH 7.4 (STE buffer) and 'free' parasites obtained by the saponin lysis method [13]. The parasite pellet (1 g wet wt.) was washed 5 times with STE buffer by centrifugation (4 200 X g, 5 min) and transferred to a small pre-chilled mortar using a minimal volume of STE buffer. The cells were mixed with approximately 2 g of washed silicon carbide to form a thin paste and the cells disrupted by grinding with a pestle. The extent of cell breakage was estimated by monitoring the release of lactate dehydrogenase from the parasites. After each 30 s period of grinding, 10 lal samples were withdrawn, diluted 10-fold with STE buffer and centrifuged ( 10 000 X g, 10 min) in an Eppendorf microcentrifuge. An aliquot of each supernatant was assayed for lactate dehydrogenase activity [14] and the percent cell breakage calculated relative to the amount released from a zero time sample treated with 0. I% (v/v, final concentration) Triton X-100. By this criterion, 50% cell disruption was obtained after grinding for 3 min. The homogenate was diluted with 20 mi STE buffer and the silicon carbide was removed by centrifugation (100 X g, 3 min). The supernatant was removed and the pellet washed with 10 ml STE buffer as before. The combined supernatants were centrifuged (1 000 X g, 10 min) to remove unbroken parasites and nuclei and the supernatant stored overnight before loading onto the magnetic separation device. All operations were at 0-4°C.
Magnetic separation apparatus. The apparatus consists of a circular C-type Alnico permanent magnet (3.7 X 11.5 X 14 cm, obtained from Magnetic Developments, P.O. Box 80, Swindon, Wiltshire, SN2 6HA, U.K.) which generates a uniform field strength of 0.7 tesla between the poles. A Perspex chamber (1.3 X 1.3 X 0.9 cm) was filled to a packing density of 7.6% with 25 tam diameter stainless steel wool and inserted between the poles of the magnet and washed with STE buffer by upward displacement to remove any air bubbles. Parasite supernatant was pumped through the chamber and the effluent collected with a fraction collector. The magnetic field from the permanent magnet induces a magnetic field in the steel wool, which then magnetically
309 binds the paramagnetic particles. After washing with buffer to remove loosely entrapped material, removal of the permanent magnet results in immediate demagnetization of the steel wool and release of the magnetically bound particles.
Determination ofmalarialpigment. Concentrations of malarial pigment were determined using a Pye Unicam SP 1800 spectrophotometer, equipped with scale expansion and zero suppression devices. The absolute absorbance spectrum was recorded for each sample in the range 600 to 700 nm and the difference in absorbance (AA) between the characteristic peak at 648 nm and trough at 618 nm was determined. Samples were generally diluted 20-fold with STE buffer and measured against a suitable scatter blank inserted in the reference position of the instrument. Pigment concentrations are expressed as nmol heme m1-1, using an extinction coefficient A~3648_618n m = 1 890 M -1 cm-L This extinction coefficient was determined by measuring heme content as pyridine hemochrome [15] in pigment purified by the method of Deegan and Maegraith [3]. Heme content was proportional to AA and A£648_618 nrn was determined by linear regression. Protein content of fractions was determined by the method of Lowry et al. [16], using bovine serum albumin as standard. RESULTS AND DISCUSSION Previous methods for the purification of pigment have followed its distribution by visual inspection. In order to quantify recoveries of hemozoin during purification, we have made use of the characteristic absorbance maximum at 648 nm, which is not found in other hemoproteins [3]. However, the absolute absorbance at 648 nm is unreliable due to light scattering, particularly in crude samples. This difficulty was circumvented by measuring the absorbance difference between peak and trough at 648 and 618 nm, respectively. This assay method has a number of advantages over other methods of measuring pigment, such as determining total heme or iron content [4,5] because it is rapid, non-destructive and unaffected by the presence of other heme-containing compounds such as oxyhemoglobin or methemoglobin. It is also accurate and reproducible, since in our attempts to purify pigment by differential centrifugation or sucrose density gradient centrifugation recoveries of 95 to 105% were routinely obtained. Malarial pigments from other Plasmodium species also possess absorbance maxima and minima in this region of the absorption spectrum [5], and this method could be adapted for use with pigment extracted from other species. A 1 000 X g supernatant was prepared from isolated P. berghei parasites which had been disrupted by grinding with silicon carbide as described in the methods. As illustrated in Fig. 1, at point A the supernatant (63.9 mg protein in total) was pumped through the apparatus and 1 ml fractions collected. Of the recovered protein, 99.5% elutes in the flow through (fractions 1- 16, inclusive) and in the subsequent washing step (point B, fractions 17-20, inclusive). No malarial pigment could be detected in these
310 A
B
C
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rr rl
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10 20 FRACTION NUMBER
!
!
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Fig. I. Purification of malarial pigment using a magnetic separation device. Commencing at arrow (A), parasite extract (23 ml, 2.5 mg protein ml-t) was pumped (1 ml rain-1) through the chamber of the magnetic separator, followed by STE buffer (B). At (C) the magnetic field was removed and the wash continued. Fractions (1.5 ml) were collected and assayed for protein (0) and malarial pigment (e). The initial specific activity (m) of the load material was 2.66 nmol mg ~ protein. fractions, as m e a s u r e d by the a b s o r b a n c e difference at 648 minus 618 nm. H o w e v e r , at a r r o w C, as soon as the c h a m b e r is r e m o v e d from between the poles o f the magnet, the m a l a r i a l p i g m e n t is released f r o m the c h a m b e r a l o n g with a small a m o u n t o f p r o t e i n (0.5% o f the total recovered). Strikingly, the specific c o n c e n t r a t i o n of the m a l a r i a l p i g m e n t (528 n m o l mg -~ p r o t e i n ) is c o n s t a n t t h r o u g h o u t fractions 21-26, indicating that each f r a c t i o n is h o m o g e n e o u s l y enriched in m a l a r i a l pigment. The overall p u r i f i c a t i o n o f this p r o c e d u r e was 200-fold (based on n m o l heme mg -~) a n d the recoveries o f m a l a r i a l p i g m e n t a n d total p r o t e i n were 48% a n d 64%, respectively. The u n r e c o v e r e d p r o t e i n is p r e s u m a b l y t r a p p e d in the interstices of the steel wool packing, as it could be r e m o v e d by v i g o r o u s flushing with buffer. In o t h e r experiments with p i g m e n t that h a d been freed f r o m its s u r r o u n d i n g m e m b r a n e , it was f o u n d that such p r e p a r a t i o n s did not elute f r o m the c h a m b e r on r e m o v a l o f the magnetic field a n d c o u l d only be released by flushing with 0.1 N N a O H . We presume, therefore, that the p i g m e n t recovered in this e x p e r i m e n t is c o n t a i n e d within m e m b r a n e vesicles. F u r t h e r studies are r e q u i r e d to test this hypothesis. The present m e t h o d of p u r i f y i n g m a l a r i a l p i g m e n t has c o n s i d e r a b l e a d v a n t a g e s over previous m e t h o d s . O t h e r studies have e m p l o y e d harsh t r e a t m e n t such as extraction with acetic acid [5] o r t r e a t m e n t with s o d i u m d o d e c y l s u l p h a t e a n d digestion with p r o t e a s e s [4]. A n a l y s e s of such p r e p a r a t i o n s have shown that all o f the iron c o n t a i n e d in p i g m e n t can be a c c o u n t e d for as hemin [4,5]. Recalculation of these d a t a indicates that h e m o z o i n f r o m P. lophurae a n d P. berghei (NS) c o n t a i n s 1 100 [5] and 200 [4] n m o l m g - ' d r y wt., respectively. The purity o f o u r p r e p a r a t i o n which c o n t a i n s 528
311 n m o l m g -~ p r o t e i n ( e q u iv a l e n t to 400 n m o l mg -x dry wt., a s s u m i n g m a l a r i a l p i g m e n t c o n t a i n s only p r o t e i n a n d heme) c o m p a r e s f a v o r a b l y to o t h er m e t h o d s for purifying pigment. This t e c h n i q u e provides a new a p p r o a c h for the isolation o f malarial p i g m e n t in a highly purified f o r m and should p r o v e useful in studies on the m o d e o f action o f a n t i m a l a r i a l s such as c h l o r o q u i n e that are t h o u g h t to interact with f e r r i p r o t o p o r p h y rin I X [17]. ACKNOWLEDGEMENTS We w o u l d like to t h a n k Mr. Stanley G r a n t for his technical assistance and Mr. Louis Schofield for carrying o u t p r e l i m i n a r y experiments on h e m o z o i n estimation. This study was f u n d e d in part by grants f r o m U N D P / W o r l d B a n k / W H O Special P r o g r a m m e for Research and T r a i n i n g in T r o p i c a l Diseases (project n u m b e r s 800033 and 820153). D C W is s u p p o r t e d by f u n d in g f r o m the Public H e a l t h L a b o r a t o r y Service, U.K. REFERENCES 1 2 3
4 5 6 7 8 9
10
11 12 13
Sherman, I.W. (1979) Biochemistry of Plasmodium (malaria parasites). Microbiol. Rev. 43,453-495. Homewood, C.A. (1978) Biochemistry. In: Rodent Malaria, (Killick-Kendrick, R. and Peters, W., eds.), pp. 169-211, Academic Press, New York, NY. Deegan, T. and Maegraith, B.G. (1956) Studies on the nature of malarial pigment (hemozoin). I. The pigment of the Simian species, Plasmodium knowlesi and P. cynomolgi. Ann. Trop. Med. Parasitol. 50, 194-211. Homewood, C.A., Moore, G.A., Warhurst, D.C. and Atkinson, E.M. (1975) Purification and some properties of malarial pigment. Ann. Trop. Med. Parasitol. 69, 283-287. Yamada, K.A. and Sherman, I.W. (1979) Plasmodium lophurae: Composition and properties of hemozoin, the malarial pigment. Exp. Parasitol. 48, 61-74. Moore, G.A. and Boothroyd, B. (1974) Direct resolution of the lattice planes of malarial pigment. Ann. Trop. Med. Parasitol. 68, 489. Chou, A.C., Chevli, R. and Fitch, C.D. (1980) Ferriprotoporphyrin IX fulfills the criteria for identification as the chloroquine receptor of malaria parasites. Biochemistry 19, 1543-1549. Orjih, A.U., Banyal, H.S., Chevli, R. and Fitch, C.D. (1981) Hemin lyses malaria parasites. Science 214, 667-669. Fitch, C.D., Chevli, R., Banyal, H.S., Phillips, G., Pfaller, A. and Krogstad, D.J. (1982) Lysis of Plasmodium falciparum by ferriprotoporphyrin IX and a chloroquine-ferriprotoporphyrin IX complex. Antimicrob. Agents Chemother. 21,819-822. Paul, F., Roath, S., Melville, D., Warhurst, D.C. and Osisanya, J.O.S. (1981) Separation of malaria-infected erythrocytes from whole blood: Use of a selectivehigh-gradient magnetic separation technique. Lancet ii, 70-71. Heidelberger, M., Mayer, M.M. and Demarest, C.R. (1946) Studies in human malaria. I. The preparation of vaccines and suspensions containing Plasmodia. J. Immunol. 52, 325-330. Fulton, J.D. and Grant, P.T. (1956) The sulphur requirements of the erythrocytic form of Plasmodium knowlesi. Biochem. J. 63,274-282. Reese, R.T., Langreth, S.G. and Trager, W. (1979) Isolation of stages of the human parasite Plasmodiumfalciparum from culture and from animal blood. Bull. W.H.O. 57, 53-61.
312 14
15 16 17
Bergmeyer, H.U. and Bernt, E. (1974) Lactate dehydrogenase: UV-assay with pyruvate and NADH. In: Methods of Enzymatic Analysis, (Bergmeyer, H.U., ed.), Vol. 2, pp. 574-579, Academic Press, New York, NY. Fuhrhop, J.-H. and Smith, K.M. (1975) In: Porphyrins and Metalloporphyrins (Smith, K.M., ed.), pp. 804-807, Elsevier, Amsterdam. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265-275. Fitch, C.D. (1983) Mode of action of antimalarial drugs. In: Malaria and the Red Cell, (Ciba Foundation Symposium 94), pp. 222-232, Pitman, London.
307
MBP 00460
A SIMPLE MAGNETIC METHOD FOR THE PURIFICATION OF MALARIAL PIGMENT
ALAN H. FAIRLAMB 1'*, FRANK PAUL 2 and DAVID C. WARHURST ~
~Department of Medical Protozoology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, and 2Tenovus Research Institute, Tremona Road, Southampton S09 4XY, England.
(Received 11 January 1984; accepted 22 March 1984)
Using a simple magnetic separation apparatus, Plasmodium berghei malarial pigment has been purified 200-fold in a single step. The technique exploits the paramagnetic properties of malarial pigment (hemozoin) and provides a simple and rapid method for the isolation of this material with high purification and
yield. Key words: Malarial pigment; Hemozoin; Plasmodium berghei; Purification; Magnetic technique
INTRODUCTION
Erythrocyte hemoglobin provides a major source of amino acids for the growth of the intracellular form of the malarial parasite. The end product of hemoglobin digestion within the food vacuole is malarial pigment or hemozoin, which can be readily seen as golden brown-black deposits in the trophozoite and schizont stages of the parasite (reviewed in [ 1,2]). After solubilization with alkali, the main constituent of malarial pigment can be isolated as hematin [3-5], but in the insoluble native state, the iron porphyrin may be associated with protein [5] to form regular crystalline arrays [6]. Malarial pigment may represent the parasites' means of sequestering free heme in an inert, non-toxic form [1]. The mechanism by which heme is complexed as malarial pigment is of interest from a chemotherapeutic standpoint, since unsequestered heine may represent the high-affinity binding site for the antimalarial drug chloroquine [7]. Moreover, the drug's antimalarial action may reside in the fact that chloroquine-ferriprotoporphyrin IX complexes lyse the malarial parasite [8,9]. High-spin hematin and consequently malarial pigment are paramagnetic, in contrast to low-spin oxyhemoglobin of the uninfected erythrocyte, which is diamag-
Abbreviation: STE buffer, 250 mM sucrose, 1 mM EDTA, 25 mM Tris-HCl, pH 7.4. *Present address: Laboratory of Medical Biochemistry, The Rockefeller University, 1230 York Avenue, New York, NY 10021, U.S.A. 0166-6851/84/$03.00 © 1984 Elsevier Science Publishers B.V.
308 netic. This difference in the magnetic properties of infected and uninfected erythrocytes has been exploited to selectively concentrate red cells infected with late-stage parasites from whole blood [10,11]. In this report, we show that this technique can be applied to the isolation of malarial pigment itself. MATERIALS AND METHODS
Preparation of parasite homogenate. Mice infected with Plasmodium berghei (N strain) 4 days previously were bled by cardiac puncture under ether anesthesia and the infected blood collected in heparinized Krebs' Ringer saline. After washing twice with Krebs' Ringer solution by centrifugation (500 X g, 10 min) the red blood cells (22% parasitemia) were passed through a CF- 11 cellulose column to remove contaminating white blood cells and platelets [ 12]. The infected blood (4 ml packed cells) was washed once in 250 mM sucrose, 1 mM E D T A , 25 mM Tris-HC1, pH 7.4 (STE buffer) and 'free' parasites obtained by the saponin lysis method [13]. The parasite pellet (1 g wet wt.) was washed 5 times with STE buffer by centrifugation (4 200 X g, 5 min) and transferred to a small pre-chilled mortar using a minimal volume of STE buffer. The cells were mixed with approximately 2 g of washed silicon carbide to form a thin paste and the cells disrupted by grinding with a pestle. The extent of cell breakage was estimated by monitoring the release of lactate dehydrogenase from the parasites. After each 30 s period of grinding, 10 lal samples were withdrawn, diluted 10-fold with STE buffer and centrifuged ( 10 000 X g, 10 min) in an Eppendorf microcentrifuge. An aliquot of each supernatant was assayed for lactate dehydrogenase activity [14] and the percent cell breakage calculated relative to the amount released from a zero time sample treated with 0. I% (v/v, final concentration) Triton X-100. By this criterion, 50% cell disruption was obtained after grinding for 3 min. The homogenate was diluted with 20 mi STE buffer and the silicon carbide was removed by centrifugation (100 X g, 3 min). The supernatant was removed and the pellet washed with 10 ml STE buffer as before. The combined supernatants were centrifuged (1 000 X g, 10 min) to remove unbroken parasites and nuclei and the supernatant stored overnight before loading onto the magnetic separation device. All operations were at 0-4°C.
Magnetic separation apparatus. The apparatus consists of a circular C-type Alnico permanent magnet (3.7 X 11.5 X 14 cm, obtained from Magnetic Developments, P.O. Box 80, Swindon, Wiltshire, SN2 6HA, U.K.) which generates a uniform field strength of 0.7 tesla between the poles. A Perspex chamber (1.3 X 1.3 X 0.9 cm) was filled to a packing density of 7.6% with 25 tam diameter stainless steel wool and inserted between the poles of the magnet and washed with STE buffer by upward displacement to remove any air bubbles. Parasite supernatant was pumped through the chamber and the effluent collected with a fraction collector. The magnetic field from the permanent magnet induces a magnetic field in the steel wool, which then magnetically
309 binds the paramagnetic particles. After washing with buffer to remove loosely entrapped material, removal of the permanent magnet results in immediate demagnetization of the steel wool and release of the magnetically bound particles.
Determination ofmalarialpigment. Concentrations of malarial pigment were determined using a Pye Unicam SP 1800 spectrophotometer, equipped with scale expansion and zero suppression devices. The absolute absorbance spectrum was recorded for each sample in the range 600 to 700 nm and the difference in absorbance (AA) between the characteristic peak at 648 nm and trough at 618 nm was determined. Samples were generally diluted 20-fold with STE buffer and measured against a suitable scatter blank inserted in the reference position of the instrument. Pigment concentrations are expressed as nmol heme m1-1, using an extinction coefficient A~3648_618n m = 1 890 M -1 cm-L This extinction coefficient was determined by measuring heme content as pyridine hemochrome [15] in pigment purified by the method of Deegan and Maegraith [3]. Heme content was proportional to AA and A£648_618 nrn was determined by linear regression. Protein content of fractions was determined by the method of Lowry et al. [16], using bovine serum albumin as standard. RESULTS AND DISCUSSION Previous methods for the purification of pigment have followed its distribution by visual inspection. In order to quantify recoveries of hemozoin during purification, we have made use of the characteristic absorbance maximum at 648 nm, which is not found in other hemoproteins [3]. However, the absolute absorbance at 648 nm is unreliable due to light scattering, particularly in crude samples. This difficulty was circumvented by measuring the absorbance difference between peak and trough at 648 and 618 nm, respectively. This assay method has a number of advantages over other methods of measuring pigment, such as determining total heme or iron content [4,5] because it is rapid, non-destructive and unaffected by the presence of other heme-containing compounds such as oxyhemoglobin or methemoglobin. It is also accurate and reproducible, since in our attempts to purify pigment by differential centrifugation or sucrose density gradient centrifugation recoveries of 95 to 105% were routinely obtained. Malarial pigments from other Plasmodium species also possess absorbance maxima and minima in this region of the absorption spectrum [5], and this method could be adapted for use with pigment extracted from other species. A 1 000 X g supernatant was prepared from isolated P. berghei parasites which had been disrupted by grinding with silicon carbide as described in the methods. As illustrated in Fig. 1, at point A the supernatant (63.9 mg protein in total) was pumped through the apparatus and 1 ml fractions collected. Of the recovered protein, 99.5% elutes in the flow through (fractions 1- 16, inclusive) and in the subsequent washing step (point B, fractions 17-20, inclusive). No malarial pigment could be detected in these
310 A
B
C
4,
;,
;
r-~
7-
tD 'o~
"7
Ee
-~2
20~
uO c
c~
E
] 600
Z
7"'']500
Z W
(D 10 h--
rr rl
d <~ .d
I
1
I
I
I
I
10 20 FRACTION NUMBER
!
!
3O
Fig. I. Purification of malarial pigment using a magnetic separation device. Commencing at arrow (A), parasite extract (23 ml, 2.5 mg protein ml-t) was pumped (1 ml rain-1) through the chamber of the magnetic separator, followed by STE buffer (B). At (C) the magnetic field was removed and the wash continued. Fractions (1.5 ml) were collected and assayed for protein (0) and malarial pigment (e). The initial specific activity (m) of the load material was 2.66 nmol mg ~ protein. fractions, as m e a s u r e d by the a b s o r b a n c e difference at 648 minus 618 nm. H o w e v e r , at a r r o w C, as soon as the c h a m b e r is r e m o v e d from between the poles o f the magnet, the m a l a r i a l p i g m e n t is released f r o m the c h a m b e r a l o n g with a small a m o u n t o f p r o t e i n (0.5% o f the total recovered). Strikingly, the specific c o n c e n t r a t i o n of the m a l a r i a l p i g m e n t (528 n m o l mg -~ p r o t e i n ) is c o n s t a n t t h r o u g h o u t fractions 21-26, indicating that each f r a c t i o n is h o m o g e n e o u s l y enriched in m a l a r i a l pigment. The overall p u r i f i c a t i o n o f this p r o c e d u r e was 200-fold (based on n m o l heme mg -~) a n d the recoveries o f m a l a r i a l p i g m e n t a n d total p r o t e i n were 48% a n d 64%, respectively. The u n r e c o v e r e d p r o t e i n is p r e s u m a b l y t r a p p e d in the interstices of the steel wool packing, as it could be r e m o v e d by v i g o r o u s flushing with buffer. In o t h e r experiments with p i g m e n t that h a d been freed f r o m its s u r r o u n d i n g m e m b r a n e , it was f o u n d that such p r e p a r a t i o n s did not elute f r o m the c h a m b e r on r e m o v a l o f the magnetic field a n d c o u l d only be released by flushing with 0.1 N N a O H . We presume, therefore, that the p i g m e n t recovered in this e x p e r i m e n t is c o n t a i n e d within m e m b r a n e vesicles. F u r t h e r studies are r e q u i r e d to test this hypothesis. The present m e t h o d of p u r i f y i n g m a l a r i a l p i g m e n t has c o n s i d e r a b l e a d v a n t a g e s over previous m e t h o d s . O t h e r studies have e m p l o y e d harsh t r e a t m e n t such as extraction with acetic acid [5] o r t r e a t m e n t with s o d i u m d o d e c y l s u l p h a t e a n d digestion with p r o t e a s e s [4]. A n a l y s e s of such p r e p a r a t i o n s have shown that all o f the iron c o n t a i n e d in p i g m e n t can be a c c o u n t e d for as hemin [4,5]. Recalculation of these d a t a indicates that h e m o z o i n f r o m P. lophurae a n d P. berghei (NS) c o n t a i n s 1 100 [5] and 200 [4] n m o l m g - ' d r y wt., respectively. The purity o f o u r p r e p a r a t i o n which c o n t a i n s 528
311 n m o l m g -~ p r o t e i n ( e q u iv a l e n t to 400 n m o l mg -x dry wt., a s s u m i n g m a l a r i a l p i g m e n t c o n t a i n s only p r o t e i n a n d heme) c o m p a r e s f a v o r a b l y to o t h er m e t h o d s for purifying pigment. This t e c h n i q u e provides a new a p p r o a c h for the isolation o f malarial p i g m e n t in a highly purified f o r m and should p r o v e useful in studies on the m o d e o f action o f a n t i m a l a r i a l s such as c h l o r o q u i n e that are t h o u g h t to interact with f e r r i p r o t o p o r p h y rin I X [17]. ACKNOWLEDGEMENTS We w o u l d like to t h a n k Mr. Stanley G r a n t for his technical assistance and Mr. Louis Schofield for carrying o u t p r e l i m i n a r y experiments on h e m o z o i n estimation. This study was f u n d e d in part by grants f r o m U N D P / W o r l d B a n k / W H O Special P r o g r a m m e for Research and T r a i n i n g in T r o p i c a l Diseases (project n u m b e r s 800033 and 820153). D C W is s u p p o r t e d by f u n d in g f r o m the Public H e a l t h L a b o r a t o r y Service, U.K. REFERENCES 1 2 3
4 5 6 7 8 9
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