Journal of Inorganic Biochemistry 104 (2010) 1119–1124
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i n o r g b i o
On the kinetics of formation of hemozoin, the malaria pigment
Robert F. Pasternack a,⁎, Ben Munda a, Abigail Bickford b, Esther J. Gibbs b, Luigi Monsù Scolaro c
a
b
c
Department of Chemistry & Biochemistry, Swarthmore College Swarthmore, PA 19081, USA
Department of Chemistry, Goucher College, Towson, MD 21204, USA
Dipartimento di Chimica Inorganica, Chimica Analitica e Chimica Fisica, C.I.R.C.M.S.B., Università di Messina, Italy
a r t i c l e
i n f o
Article history:
Received 24 February 2010
Received in revised form 30 June 2010
Accepted 30 June 2010
Available online 29 July 2010
a b s t r a c t
We report on the kinetics of formation of hemozoin as a function of hemin concentration and reaction
medium. Evidence is presented for the critical role played by interfacial regions in the efficient conversion of
hemin to the malaria pigment.
© 2010 Elsevier Inc. All rights reserved.
Keywords:
Malaria
Hemozoin
Chloroquine
Heme
Hemin
1. Introduction
Despite years of intensive study malaria remains one of mankind's
most dreaded scourges with annual infections and deaths counted in
the hundreds of millions [1]. A critical factor in the expression of the
disease is a pathway by which the Plasmodium parasite protects itself
from the presence of extra-protein heme. As the parasite feeds on the
protein portion of hemoglobin, large amounts of toxic heme are
released. The heme is rapidly oxidized to hemin (iron in the + 3
oxidation state) which, in a series of steps, is converted to and stored
as an insoluble, non-toxic, largely unreactive assembly called
hemozoin (“malaria pigment”) [2,3]. It is generally believed that
quinine and similar drugs owe their efficacy to interference with
hemozoin formation [4], but some considerable uncertainty remains
as to the molecular details of this inhibition [5].
Although studied for nearly three centuries, the composition and
structure of hemozoin were elucidated only within the last twenty
years [6,7]; and the mechanism of its formation remains an open
question. The basic unit of the assembly is a reciprocal dimer in which a
propionate group of one hemin occupies an axial position on the iron
of a neighboring hemin, and vice versa [6]. The iron(III) centers have
been shown to be high-spin [8]. Whereas hemin is known to
aggregate extensively in aqueous solution, the “normal” dimer
involves either stacking interactions or the formation of a μ-oxo
linkage between iron centers [9–11]. Some controversy exists as to
⁎ Corresponding author. Department of Chemistry and Biochemistry, Swarthmore
College, 500 College Avenue, Swarthmore, PA 19081, USA. Tel.: + 1 610 3288559; fax:
+ 1 610 3287355.
E-mail address: rpaster1@swarthmore.edu (R.F. Pasternack).
0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.07.001
which of these forms is prevalent under the conditions of interest here
(pH ~ 5, vide infra); recent evidence indicates that the dimer structure
is highly dependent on pH and solvent composition [9,10,12,13].
What is unequivocal is that under these pH conditions, hemin is
sparingly soluble in aqueous media and precipitates as α-hematin
aggregates. This material is readily converted back to a soluble form
by modifying the pH or by treatment with any of a number of reagents
including DMSO, bicarbonate or pyridine. In contrast, the insoluble
aggregate formed by Plasmodium based on a reciprocal dimer is
resistant to attack by most reagents (including those listed above) and
to changes in pH over an extended range. This so-called β-hematin
aggregate or hemozoin involves hydrogen bonding between adjacent
dimer units for which the non-liganded propionate groupings play a
key role [7]. It is in this form that Plasmodium stores released heme [14].
The formation of hemozoin occurs in a food vacuole produced by
the parasite for protease “digestion” of hemoglobin [15]. The optimum
pH for this protein degradation activity – the pH at which hemozoin is
formed – is about five, and the half-life for the conversion of free
hemin into hemozoin has been estimated as in the order of minutes
[16]. To simulate in vivo conditions, most laboratory studies on the
kinetics of hemozoin formation are carried out in the presence of
acetate or citrate buffer to maintain the physiologically relevant pH. In
one such protocol [17], hemin in DMSO was added to an aqueous
solution containing acetate and a water-soluble alcohol. After 16 h the
reaction mixture was tested for the presence of hemozoin. A
correlation was found between the yield of the malaria pigment and
the lipophilicity of the dissolved alcohol.
A second protocol to study hemozoin formation, developed by
Egan and coworkers [4,18], uses a closed system reactor in which a
large amount of hemin (75 mg) is placed in about 25 mL of 4.5 M
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R.F. Pasternack et al. / Journal of Inorganic Biochemistry 104 (2010) 1119–1124
acetate buffer and maintained at 60 °C (the reaction is extremely slow
at physiological temperature). Under these conditions hemin rapidly
aggregates and precipitates in the α-hematin form. Samples are
withdrawn at intervals over the course of many hours and are cooled
on ice, dried for 48 h and eventually studied via infrared spectroscopy
to determine the extent of conversion to β-hematin. This protocol has
been critiqued by its originators who pointed out the non-correspondence of the conditions (especially temperature and method of hemin
delivery) to those found in vivo [16]. In still another protocol
developed by this group a small sample of a hemin stock in aqueous
acetone is injected at the interface of a citrate containing aqueous/
organic mixture [16]. Using this approach, efficient conversion to
hemozoin was observed in the presence of several lipids and two
alcohols (1-pentanol and 1-octanol).
We describe below an alternative, complementary method for
studying the kinetics of hemozoin formation, which we believe has
several advantages; it is more flexible than previous protocols and
lends itself to systematic kinetic studies. We compare the results
obtained using this new protocol to those already reported, and
discuss factors that appear vital for rapid, efficient conversion of
hemin into hemozoin.
2. Experimental
2.1. Materials and methods
Fluka and Frontier Scientific hemin were used for these experiments. Chloroquine (ChlQ) diphosphate was a MP Biomedicals
product, and L-α-Dioleoylphosphatidyl-ethanolamine (DOPE) (N99%
purity) was supplied by Avanti Polar Lipids, Inc. (Alabama). Other
more standard chemicals were obtained from Fisher Scientific and
Aldrich Chemicals and met ACS standards for high purity.
Reaction vials (8 mL) fitted with teflon caps were used as reaction
vessels. These were maintained at a constant temperature using an
Isotemp 202 S bath supplied by Fisher Scientific. The vortex devices
employed were a Thermolyne MaxiMix II and a Scientific Industry
Vortex-2 Genie. Centrifugations were performed on a Sorvall Instruments RC5C fitted with a SS-34 head. Spectroscopic measurements
were conducted on a JASCO V-560 spectrophotometer.
of the lipid is added to this stock. This mixture is monophasic
for all but one of the co-solvents we have tested and report on
below (heptane). In the heptane case, the organic mixture and
acetate solutions were added separately to the reaction vials. In
the standard protocol, 250 μL of Stock D is added to each of a
selected number of screw-top reaction vials (usually 15–20)
that are then equilibrated to 37 °C.
(iii) To initiate the reaction, 750 μL of the thermally equilibrated
hemin (Stock C) is added to each vial. The reaction mixture is
inverted twice and immediately returned to the constant
temperature bath. In an earlier study XRD, FTIR and Resonance
Raman spectroscopies were used to show that hemin produces
hemozoin in such media [16,21].
Under these conditions, depending on the co-solvent (or
presence of a lipid), the hemin either rapidly precipitates (as
α-hematin) and/or is selectively taken up in the co-solvent that
has formed a separate phase upon the addition of the aqueous
hemin stock. The co-solvent represents about 2% of the total
reaction volume, and the recorded pH* of the reaction mixture
was 5.3.
(iv) At selected times, reaction vials are removed from the bath and
plunged into ice. As quickly as possible, 5 mL of a solvent
system containing 50% acetone/aqueous pyridine (5%) buffered
with 26 mM HEPES is added. The resulting mixture is vortexed
for 60 s to ensure that the pyridine has encountered all the
hemin regardless of its state. At this point, all hemin that is not
part of a hemozoin assembly is converted to soluble hemin
(py)2. This assay solution was shown – through application of
X-ray diffraction and infrared spectroscopy – not to react with
hemozoin but to readily dissolve hemin and α-hematin and
convert them to the low-spin dipyridine hemin complex [21].
(v) The mixture is centrifuged for 30 min at 5000 rpm to separate
the insoluble hemozoin which settles to the bottom of the vial.
The supernatant is then tested spectrophotometrically (λmax at
406 nm) for the presence of hemin as the dipyridine adduct.
The data is plotted (Figs. 1–5) as the percentage of hemin
converted to hemozoin (% conversion) vs. time.
2.2. Protocol
A new protocol used for studying the kinetics of hemozoin
formation involves several steps:
(i) Prepare a concentrated solution (~ 10–20 mM) of hemin in
DMSO (Stock A). Hemin is very soluble in this solvent and
exists as a monomer [19]. To keep the DMSO concentration
below 0.5% in the final reaction mixture, it proves convenient to
prepare Stock B in aqueous 1 mM phosphate buffer (final
pH ~ 10) by a dilution of Stock A by 20 fold. The concentration of
Stock B is determined spectrophotometrically using a sample
diluted by a factor of one hundred with a 0.1 M NaOH solution.
The molar absorptivity of hemin in this medium has been
reported as ε = 5.84 × 104 M− 1 cm− 1 [20]. Finally, a large
volume (~20 mL) of a hemin reaction solution (Stock C) in
1 mM phosphate buffer (pH ~ 10) is prepared from Stock B such
that hemin in the reaction vials will have the desired final
concentration (50 μM for most of these experiments). Stock C,
which is monophasic, is equilibrated at 37o in a constant
temperature bath. When diluted by a factor of one-third (vide
infra), DMSO is less than 0.5% by volume in the reaction
mixture.
(ii) Another stock solution (Stock D) is prepared which typically
contains 9 mL methanol, 1 mL of co-solvent, and 2.5 mL of 1 M
acetate buffer, pH ~ 5. For lipid experiments, a weighed quantity
Fig. 1. Kinetic profiles for the conversion of hemin into hemozoin for several co-solvents
listed in Table 1. Although the co-solvents represent only about 2% of the reaction
volume, they can have a profound impact on the kinetics of conversion. The curves
shown represent the best fit of the data obtained by applying Eq. (1). For 1-hexanol,
k = 0.14 min− 1, n = 1.3, ks = 0.0054 min− 1; while for CCl4, k = 0.012 min− 1 and
n = 0.20. The formation of hemozoin in a heptane/methanol/aqueous medium
represents less than 10% conversion over the time window studied (~200 min).
R.F. Pasternack et al. / Journal of Inorganic Biochemistry 104 (2010) 1119–1124
Fig. 2. Comparison of the kinetics of hemozoin production in the presence of several
straight chain alcohols. Under the conditions of the experiments (9:1 methanol to cosolvent) both 1-hexanol and 1-octanol form a separate phase when aqueous hemin is
added while 1-pentanol does not.
2.3. Kinetic analysis
The kinetic data obtained for a number of the systems studied here
are biphasic (cf. Fig. 1, 1-hexanol) having a major absorbance change
that is quite rapid (comparable to the half-life estimated for in vivo
formation of hemozoin), and a smaller, slower effect whose half-life is
measured in hours. The data can be fit as two parallel processes in
which the primary change is represented by a “stretched exponential”
(Avrami equation [22]) and the slower process (for which less precise
data is available) by a simple exponential. The fit to the data for 1hexanol in Fig. 1, as well as several other co-solvents, was obtained
using the working equation:
n + 1
y = ð1−A∞ = Ao Þ100−y1 exp −ðkt Þ
= n + 1 −y2 expð−ks t Þ ð1Þ
where “y” is the total % Conversion of hemin to hemozoin at the time, t,
as calculated from absorbance measurements at 406 nm for the
dipyridine hemin complex, and “y1” and “y2” are the contributions
made to the conversion via the more rapid and slower processes. A∞
and Ao represent the final and initial absorbances at 406, respectively.
After 24 h, the absorbance at 406 nm approaches zero for the more
active solvent systems investigated.
Fig. 3. Study of the impact of an aqueous phase on the conversion of hemin to hemozoin
in the presence of 1-octanol as co-solvent.
1121
Fig. 4. Effect of added lipid (DOPE) on the kinetics of hemozoin formation. The
methanol/chloroform/aqueous system is among the least active in producing
hemozoin, in contrast to its reactivity with lipid added.
In a kinetic model developed earlier for autocatalytic processes
[23,24], a key step involves the rate-determining formation of a “seed”
or “nucleus” catalyzed by a (fractal) surface. In the limiting case in
which the “seed” size is one (that is, the rate-determining step involves a
solute species undergoing a transformation rather than the formation of a
multi-molecular seed), the resulting integrated rate law has a “stretched
exponential” functional form as in the first term of Eq. (1).
We use this semi-empirical approach to compare and contrast
reaction systems differing in co-solvent, added lipid and/or added
chloroquine. For the results shown in Fig. 1, we obtain: for 1-hexanol
as co-solvent, k = 0.14 min− 1, n = 1.3, and ks = 0.0054 min− 1; for
carbon tetrachloride as co-solvent, k = 0.012 min− 1 and n = 0.20. To
facilitate comparisons of kinetic results obtained under a variety of
conditions, we introduce estimates of the initial half-life (t1/2′) for the
stretched exponential function (t1/2′ = {[0.693 (n + 1)]^1/(n + 1)}/k),
and for the slow (simple exponential) process (t1/2 s = 0.693/ks).
These calculations are based on the approximation that the two
kinetic processes are sufficiently different in rate that they can be
effectively decoupled. For the results shown in Fig. 1, t1/2′ = 8.7 min
and t1/2 s = 2.1 h for 1-hexanol; t1/2′ = 71 min for CCl4.
Fig. 5. Influence of added chloroquine (ChlQ) on the formation of hemozoin with 1octanol as co-solvent. The ChlQ has a marked inhibitory effect, which scales with its
concentration.
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R.F. Pasternack et al. / Journal of Inorganic Biochemistry 104 (2010) 1119–1124
3. Results
Table 2
Summary of kinetic resultsa.
3.1. Survey of co-solvents
System
Experiments were conducted using the protocol described above
that differed from one another in the identity of the co-solvent —
which comprises about 2% by volume of the final reaction mixture.
Reagents were selected having various functional groups and
lipophilicities, and the results were used as a method of screening
the several organic solvents for their impact on hemozoin production.
The criterion that was applied was the percentage conversion after
100 min of reaction time. A summary of the results is shown in
Table 1. Fuller kinetic studies were conducted for those co-solvents
that are particularly active.
One of the active alcohols (1-octanol) was selected as co-solvent to
probe the impact on the kinetics of hemin and DMSO concentration.
Three experiments were conducted: [hemin]= 50 μM, DMSO ~ 0.3% by
volume; [hemin]= 50 μM, DMSO ~ 5% and [hemin]= 400 μM, DMSO
~5%. The kinetic profiles obtained for these experiments are nearly
indistinguishable with k = 0.16 min− 1, n = 0.83; ks = 0.0066 min− 1;
k = 0.16 min− 1, n = 0.93, ks = 0.0060 min− 1; and k = 0.17 min− 1,
n = 0.79, ks = 0.0082 min− 1, respectively (Table 2).
Kinetic profiles for three straight chain alcohols, 1-octanol, 1hexanol and 1-pentanol, are shown in Fig. 2. Both the octanol and
hexanol systems exhibit phase separation upon addition of the
aqueous hemin solution (Step (iii) of the Protocol) and the hemin is
concentrated in the now separated co-solvent volume (although
some color remains in the major aqueous/methanol phase).
However, 1-pentanol under these conditions shows no such phase
separation. Note how different the 1-pentanol kinetic profile is from
the other alcohols. Values of t1/2′ are 110 min for 1-pentanol and only
7–9 min for the other two alcohols. If the experimental conditions
are modified somewhat (Step ii) so that the organic mixture is now
6:4 methanol to 1-pentanol, an organic phase does separate on
aqueous hemin addition, and the 1-pentanol profile is now very
similar to the other alcohols. Under these conditions of 6:4 methanol
to alcohol, t1/2′ for 1-octanol and 1-pentanol are 12 and 10 min,
respectively.
The importance of interface regions for rapid kinetics is further
illustrated by an experiment in which Stock D (methanol with 1octanol) was prepared so that it did not contain acetate buffer. The
Step (iii) addition was thus divided into two parts. First, 50 μL of an
aqueous solution of 1 M acetate buffer was added to 750 μL hemin
Stock C in each room temperature reaction vial, leading to rapid
precipitation of α-hematin at pH ~ 5. After several minutes, these
mixtures were centrifuged and the near-colorless supernatant was
discarded. Next, some 200 μL of the pre-equilibrated organic mixture,
9:1 methanol/1-octanol, was added to the solid material remaining in
Table 1
Comparison of organic co-solvents.
a
Co-solvent
% Conversion in 100 min
logPa
Acetone
Aniline
Carbon tetrachloride
Chloroform
Cyclohexanol
Cyclopentanone
Dichloromethane
Heptane
1-Hexanol
Nitromethane
1-Octanol
1-Pentanol
b5
b5
~ 30
b5
b5
b5
b5
b5
~ 85
b5
~ 80
~ 20
−0.157
0.936
2.861
1.755
1.341
0.198
1.187
4.475
1.938
−0.195
3.001
1.407
logP are partition values obtained by Advanced Chemistry Development. The larger the
value, the more lipophilic is the solvent.
i) 50 μM hemin
9:1 methanol/CCl4
9:1 methanol/1-hexanol
(Figs. 1 and 2)
9:1 methanol/1-octanol
(Fig. 2)
9:1 methanol/1-octanol
(5% DMSO)
6:4 methanol/1-octanol
9:1 methanol/1-pentanol
(Fig. 2)
6:4 methanol/1-pentanol
9:1 methanol/chloroform
(DOPE added) (Fig. 4)
9:1 methanol/1-octanol
(0.1 mM chloroquine, Fig. 5)
ii) 400 μM hemin
9:1 methanol/1-octanol (5% DMSO)
n
k
(min− 1)
t1/2′
(min)
t1/2 s
(h)
0.20
1.3
0.012
0.14
71
8.7
–
2.1
0.83
0.16
7.1
1.8
0.93
0.16
7.3
1.9
3.2
11
0.11
0.011
12
110
1.6
–
1.4
0.33
0.13
0.30
10
3.1
0.83
1.3
9.1
0.022
55
2.2
0.79
0.17
6.6
1.4
a
All systems are 0.3–0.5% DMSO unless otherwise specified.
the vial, and the mixture was vortexed and maintained at 37 °C. The
usual detection procedure was then followed — vials were removed
periodically, the reaction was quenched on ice, and buffered pyridine
solution was added, followed by vortexing, centrifugation and
spectroscopic determination of conversion to hemozoin. The results
are shown in Fig. 3; the conversion to hemozoin in the absence of an
aqueous phase proceeds very slowly. The data in the absence of an
aqueous phase is fit as a first-order reaction with a rate constant of
0.0088 min− 1.
3.2. Experiments with a lipid (DOPE)
It is widely agreed that lipids play a critical role in the formation
of hemozoin in vivo [25–28]. Lipid structures have been observed
within the Plasmodium food vacuole in which hemozoin is
completely embedded (so-called “lipid nanospheres”). These lipid
droplets putatively catalyze the formation of hemozoin by concentrating the extra-protein hemin and excluding water to facilitate the
formation of the reciprocal dimer. In the current study we
considered the influence of DOPE on hemozoin production by
modifying the present protocol so that solid lipid was dissolved in
Stock D (0.50 mg/mL). When aqueous hemin is added (Step iii), the
mixture becomes extremely turbid as DOPE separates out of
solution. The lipid has a remarkable effect when CHCl3 was used as
the co-solvent (Fig. 4) giving results (k = 0.30 min− 1; n = 0.33,
ks = 0.0088 min− 1) that are rapid compared even to 1-hexanol or 1octanol as co-solvent; in the absence of the lipid, CHCl3 produces
practically no hemozoin (Table 1 and Fig. 4). By way of contrast, the
addition of DOPE to the methanol/1-octanol system has little effect
on the rate of hemozoin formation.
3.3. Chloroquine experiments
Finally, some preliminary experiments were conducted in which
chloroquine was incubated with hemin before addition to a methanol/
1-octanol/acetate buffer solution. The results are summarized in Fig. 5.
The droplet of 1-octanol which forms on aqueous hemin addition is
intensely colored as in the absence of chloroquine. Yet, the inhibition of
hemozoin formation is virtually complete at a chloroquine concentration of 1 mM, but is only partial at [ChlQ]= 0.1 mM. Under these latter
conditions, k = 0.022 min− 1, n = 9.1, ks = 0.0052 min− 1. These effects
due to ChlQ are not the result of pH changes; pH* = 5.3 for all three
reaction mixtures.
R.F. Pasternack et al. / Journal of Inorganic Biochemistry 104 (2010) 1119–1124
4. Discussion
Results described here parallel observations reported for the
formation of hemozoin in the intact biological system. At a pH of
about 5 at 37 °C, the half-life for the primary pathway to hemozoin
formation is of the order of a few minutes, lipids greatly enhance the rate
of the process and chloroquine interferes in a concentration-dependent
manner. Although conversion of hemin/α-hematin into a hemozoin
assembly is fostered by a lipophilic environment, the dependence is not
straightforward. Both the most and least lipophilic co-solvents studied
produce very little hemozoin over the time frame of interest (Table 1).
Our results point to an important role played by interface regions
between aqueous and non-aqueous phases. The slow kinetics observed
in a non-aqueous methanol/1-octanol environment (Fig. 3) is a case in
point. The reaction profile in this medium is fit as first-order with a halflife of nearly one and a half hours. For several other systems we observe
such slow (usually minor) processes, which we interpret as arising from
conversion to hemozoin within a single phase rather than at the
interface. Recall that in the method of Huy et al. [17] hemin, delivered in
DMSO to an aqueous phase containing a water-soluble alcohol, produces
hemozoin (as determined after 16 h) with an efficiency of conversion
that correlates with the lipophilicity of the alcohol. Preferential solvation
of hemin may be an important factor in this procedure. (Such
preferential solvation of water-soluble metalloporphyrins by organic
solvents has been previously reported [29]). One can envision a de facto
separation in which hemin is partially screened from water by the
alcohol; the more lipophilic the alcohol, the better is the screening. In a
slightly modified Huy procedure, when non-water-soluble, longer chain
alcohols are employed, the conversion is very much more rapid.
Further insight into the importance of interface regions emerges
from a comparison of results obtained here to ones reported by Egan et
al. [16]. The Egan procedure involves the layering of an organic solvent
on an aqueous phase (cf. Introduction). For 1-octanol, results obtained
with our protocol are remarkably similar to those reported by Egan et al.
Using a first-order rate law, they obtained a value for k of 0.13 min− 1,
while we obtain 0.16 min− 1 (Fig. 2). However, in the solvent separated
method of Egan et al., 1-pentanol was found to be very effective at
producing hemozoin from aqueous acetone-containing hemin, giving
results quite similar to 1-octanol. In contrast, we find using our protocol
that 1-pentanol as co-solvent (regardless of whether we combine it
with methanol or acetone) leads to a kinetic profile which involves an
extended incubation period; quite different from that obtained for 1octanol (Fig. 2). This difference in behavior, we suggest, is the
consequence of 9:1 methanol:1-pentanol and acetone:1-pentanol
media not separating into distinct phases when aqueous hemin is
added; the liquids are miscible under these conditions. We repeated
this study using other ratios of methanol to 1-pentanol, 1:9; 4:6; 6:4. In
all these three cases, 1-pentanol forms a discrete phase when aqueous
hemin is added, and in all three cases hemozoin is produced at rates
comparable to that observed with 1-octanol, t1/2′ = 10 and 12 min,
respectively in a 6:4 mixture. Taken together, these results provide
direct evidence for the importance of an interface region for rapid
hemozoin formation. However, notice should be taken that the
existence of an interface is not sufficient to insure efficient conversion
of hemin into hemozoin. Heptane forms an interface with aqueous
media but it is among the least effective co-solvents at producing
hemozoin.
The role of neutral lipids in producing hemozoin is illustrated by
the impact of added DOPE to a methanol/CHCl3 solvent system. The
methanol/CHCl3 system is among the least efficient of those studied
at producing hemozoin, but when the lipid is added the rate of
conversion to hemozoin is comparable to or even faster than that
observed for methanol/1-hexanol or methanol/1-octanol (cf.
Table 2). We believe that structured interface regions serve as
reaction sites that are critical for rapid hemozoin formation. A recent
study [30] using Transmission Electron Microscopy has shown that
1123
emulsions form near a lipid/aqueous interface and that hemozoin is
produced preferentially at the surface of the emulsion droplets.
Even in the presence of such interface reaction sites, chloroquine is
capable of inhibiting the formation of hemozoin. It is apparent in the
methanol/1-octanol system where direct visual detection is possible,
that hemin is still concentrated in the lipophilic phase in the presence
of chloroquine but virtually no reaction is observed at 1 mM
chloroquine. A lower concentration of chloroquine effectively blocks
formation of hemozoin for a period of time, but then an efficient
conversion occurs (Fig. 5). The profile suggests a mechanism in which
chloroquine interferes with the formation of microcrystallites which
serve as templates for hemozoin crystallization. Once such microcrystallites form, hemozoin crystals are rapidly produced.
Various step-wise chemical models have been proposed to account
for the stretched exponential kinetic profile employed in this study.
However, in most of these a concentration dependence is predicted
[31], but not observed in the present system. These models have in
common an autocatalytic process in which each molecule of product
serves as a catalytic site. In an approach previously suggested by us for
DNA-bound porphyrin assembly [23,24], the catalyst is a (fractal)
surface rather than individual product molecules. The resulting
integrated rate law shows no concentration dependence for the case
in which the rate-determining step involves the transformation of a
solute species. We propose that the key step in the mechanism of
hemozoin formation occurs at the aqueous/non-aqueous interface
where hemin exists as a dimer, and that the interface helps promote
the conversion of the standard dimer to the reciprocal dimer. Once
formed, other dimers attack the reciprocal dimer, are transformed,
and add to it (via hydrogen bonding) to form the hemozoin crystal.
Hemin exists as a monomer in organic layers and α-hematin
assemblies in aqueous phase (pH ~ 5) — and both of these forms are
slow to convert to reciprocal dimer assemblies. Thus, one way to affect
the kinetics of the process is by modifying the interface region rather
than changing the hemin concentration. It has been reported that the
rate of hemozoin production in stirred solutions depends on the rate
of stirring [18] — such stirring may well have an impact on the
properties of the interface region. That the existence and properties of
the interface have such a profound effect on the kinetics of hemozoin
production leads to a consideration of an alternative mechanism for
chloroquine activity. Previous workers have suggested that chloroquine acts by binding either to monomers or dimers of hemin or arises
from a capping of hemozoin microcrystals [4,5]. However, it is
possible that chloroquine and similar drugs show activity as a result of
their impact on the aqueous/non-aqueous (lipid) interface so as to
inhibit reaction. Further studies on this suggested inhibition mechanism are currently underway.
Abbreviations
ChlQ
Chloroquine
DOPE
L-α-Dioleoylphosphatidyl-ethanolamine
pH*
measured pH in a mixed solvent medium
t1/2',t1/2 s estimated first half-life for the stretched exponential kinetic
process and half-life for the slower, first-order process
HEPES
2-[4-(2-hydroxyethyl)-2,3,5,6-tetrahydropyrazin-1-yl]
ethanesulfonate.
Acknowledgement
We are grateful to Dr. Giovanna De Luca for aiding in the
preparation of this manuscript.
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