From the Biophysics Section, Blackett Laboratory, Imperial College of Science, Technology, and Medicine, London SW7 2BW, United Kingdom and the ¶ School of Biological Sciences, University of Southampton, Biomedical Sciences Building, Bassett, Crescent East, Southampton SO16 7PX, United Kingdom
Received for publication, January 22, 2001, and in revised form, April 2, 2001
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ABSTRACT |
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Human serum albumin (HSA) is an abundant
transport protein found in plasma that binds a wide variety of drugs in
two primary binding sites (I and II) and can have a significant impact
on their pharmacokinetics. We have determined the crystal structures at
2.5 Å-resolution of HSA-myristate complexed with the R-(+) and S-( Human serum albumin
(HSA)1 is the major protein
component of blood plasma but is also distributed to the interstitial
fluid of the body tissues. The protein binds a number of relatively insoluble endogenous compounds such as unesterified fatty acids, bilirubin, and bile acids and thus facilitates their transport throughout the circulation (1, 2). HSA is also capable of binding a
wide variety of drugs (1-3), and much of the interest in this abundant
protein derives from its effects on drug delivery. Drug binding to
plasma proteins such as HSA can be an important determinant of
pharmacokinetics, restricting the unbound concentration and affecting
distribution and elimination. In some cases the major fraction of the
administered drug is sequestered by HSA; this is particularly true of
warfarin, a widely used anticoagulant, which is 99% bound to the
protein under normal therapeutic conditions and consequently has a
small volume of distribution and low clearance (4).
HSA has a limited number of binding sites for endogenous and exogenous
ligands, so that drug binding to the protein may be affected by a
variety of factors. Although the effects on pharmacokinetics of
drug-drug competition for the same sites on HSA are generally held to
be of little clinical import (4, 5), physiological or diseased states
that cause variations in the plasma levels of albumin or its primary
endogenous ligands can influence drug binding and may require dosages
to be closely monitored. Added to this, genetic polymorphisms in HSA
can also alter drug binding and may further complicate the clinical
picture (6, 7). To understand the molecular basis of these effects,
structural information is required to fully delineate the binding sites
for drugs and endogenous ligands. Such information will also be
invaluable to efforts to exploit the carrier properties of HSA in the
development of novel therapeutic reagents for drug targeting (8) or
oxygen transport (9).
HSA, a 585-residue protein, is monomeric but contains three
structurally similar Crystallographic studies located drug sites I and II in subdomains IIA
and IIIA, respectively, and provided the first detailed description of
the geometry and chemistry of the binding environment (10). Several
reports have analyzed structural aspects of the binding of an aspirin
analogue, 2,3,5-triiodobenzoic acid (TIB), to defatted HSA (10), to the
HSA-myristate complex (11), or to equine serum albumin (19). However,
these studies were done at fairly modest resolutions (3.0-4.0 Å), and
in any case it is not clear whether TIB, which carries three iodine
atoms, is properly representative of typical HSA drug ligands.
The binding of warfarin to HSA and the interactions of the drug
with other HSA ligands have been investigated for many years. Warfarin
(3-( Purification, Crystallization, and Drug Soaking--
Recombinant
HSA, produced in yeast and supplied by Delta Biotechnology Ltd.
(Nottingham, UK), was purified by gel filtration, complexed with excess
myristic acid, and crystallized as described previously (11, 12).
Racemic warfarin, purchased from Sigma-Aldrich (A-2250) was separated
into R-(+) and S-( Data Collection and Structure Determination--
X-ray
diffraction data were collected at room temperature at station X-11 of
the European Molecular Biology Laboratory Outstation at DESY,
Hamburg, Germany (see Table I). The data were indexed and measured
with MOSFLM.2 For
both enantiomers, crystals of the HSA-myristate-warfarin complex
were isomorphous with the HSA-myristate complex (11, 12). The protein
model for this structure, stripped of its ligands, was used for initial
phasing of the x-ray data. The model, split into its six subdomains,
was initially refined using a rigid body protocol in CNS (27)
and then subjected to cycles of positional and B-factor refinement
before calculation of initial Fo Structure Determination--
HSA-myristate complexes were prepared
and crystallized as described previously (11, 12, 30). Over a period of
at least 48 h the crystals were soaked in increasing
concentrations of the warfarin R-(+) and S-( Structure of the Complex--
The overall structures of the
HSA-myristate-warfarin complexes are very similar to those reported for
HSA-myristate and other HSA-fatty acid complexes (11, 12); there are
only minor conformational changes in a number of side chains located at
the drug binding site. As expected, warfarin binds within subdomain IIA
in Sudlow's drug site I (Fig.
3A) (10, 13, 32) and displaces
the myristate molecule that binds to this domain (in fatty acid site 7, FA7) with relatively low affinity (12). In addition to the drug bound in IIA, there is a fragment of difference density adjacent to the
myristate bound to subdomain IB at the same location observed for
triiodobenzoic acid (11). The density would be large enough to
encompass the coumarin moiety of warfarin and may represent a secondary
binding site for the drug, but there is no density for the other
portions of the drug, and a second ligand molecule has therefore not
been incorporated into the model.
The Warfarin Binding Pocket--
The binding pocket is formed by
the packing of all six helices of subdomain IIA (Fig. 3C).
The R-(+) and S-(
Given the ability of HSA to accommodate a wide range of different drug
compounds in site I, it is perhaps surprising to find that warfarin
fits quite snugly into the binding pocket (Fig. 3C). The
binding site has two sub-chambers that accommodate different portions
of the warfarin molecule. The interaction between drug and protein
appears to be dominated by hydrophobic contacts, but there are also a
number of specific electrostatic interactions. The benzyl moiety binds
in a sub-pocket formed by Phe-211, Trp-214, Leu-219, and Leu-238
with additional aliphatic contacts from Arg-218 and His-242 (Fig.
3B). Direct contact between the benzyl ring and the side
chain of Trp-214 explains why modification of this residue reduces
warfarin binding to HSA (33). The proximity of the benzyl ring to
Arg-218 also accounts for the reduction in binding affinity associated
with mutations at this position (6). The coumarin moiety fits into the
main chamber furthest from the entrance, which is the same portion of
site I that is occupied by TIB (11). Fig. 3B shows that this
chamber has an additional side pocket (delimited by Leu-219, Arg-222,
Phe-223, Leu-234, Ile-264, Leu-257, and Ile-290) that is not occupied
by warfarin but may accommodate hydrophobic portions of other site I
drugs. The coumarin group makes primarily hydrophobic contacts with the surrounding side chains (Tables
II and
III and Fig. 3C). The roof and
floor of the pocket are delimited by Ala-291 and Leu-238, respectively;
the back end of the coumarin ring contacts Ile-260, Ile-264, Ile-290,
and the aliphatic portions of Arg-257 and Ser-287 and lies close to but
does not directly contact the fatty acid in the adjacent binding site
(FA2). According to the view in Fig. 3C, there are further
hydrophobic contacts on the right flank from Val-241 and on the left
from the aliphatic portion of Arg-222. In addition, two of the
three oxygen atoms contribute to electrostatic interactions. The O4
atom of the hydroxyl group makes hydrogen bonds to the side chain of
His-242 (2.9 Å) and to a bound water molecule (2.8 Å). On the other
side of the drug, although the O1 atom faces a gap in the wall of the
binding pocket and has no specific interaction with the protein, the O2
atom is positioned 3.7 Å from N Drug binding to HSA has been extensively investigated over the
past 30 years. Much of this work has been performed using binding and
competition assays to map out the number and selectivity of binding
sites on the protein (13, 16-18, 34), although more recently,
mutagenesis techniques have been applied to the study of drug
interactions (6, 35). Although initial crystallographic studies at
moderate resolution have focused mainly on the binding of TIB, a drug
analogue, to HSA (10, 11), there has been a lack of high resolution
structural information with which to interpret the amassed biochemical
and biophysical data on HSA-drug interactions.
The structures reported in this paper provide the first high resolution
view of what might reasonably be termed a "classic" site I drug
bound to HSA. Although many different drugs bind to site I, they are
generally bulky heterocyclic compounds with a negative charge located
toward the center of the molecule (34). Warfarin exemplifies this type
of compound, and the structure of the HSA-myristate-warfarin complex
shows how this broad selectivity is achieved. The coumarin and benzyl
moieties are accommodated in separate but adjacent chambers of what has
previously been described as a "sock-shaped" pocket (10). The polar
features of the drug are all found at the basic mouth of the pocket,
with the exception of the hydroxyl oxygen (O4), which interacts
specifically with the side chain of His-242 on one wall of the pocket
(Fig. 3C).
Both enantiomers of warfarin bind in essentially the same way to the
protein. This accounts for the poor stereoselectivity of albumin and
suggests that the prospects for developing it as a reagent for chiral
separations of warfarin are unfavorable. Conversely, the structure
clearly reveals the details of the HSA-warfarin interaction and
provides a structural basis for the possible design of warfarin
derivatives with altered HSA binding properties. The binding of several
warfarin derivatives to HSA has already been characterized;
acenocoumarin binds more loosely than warfarin, and phenprocoumon binds
more tightly (16, 22, 34). The weaker association of acenocoumarin
probably occurs because the addition of an NO2 group
to the benzyl ring sterically hinders its accommodation in the
hydrophobic compartment formed primarily by Phe-211, Trp-214, Leu-219,
and Leu-238. By contrast the tighter binding of phenprocoumon, in which
the CH2-CO-CH3 acetonyl group found on warfarin
is replaced by a propyl moiety (C3H7), may be
due to the formation of hydrophobic contacts between this propyl group
and the side chains of Trp-214 and Arg-218 (Fig. 3C).
Comparison with the structure of HSA-myristate-TIB (11) reveals
differences in the ways that TIB and warfarin bind to site I. The
heterocyclic coumarin moiety of warfarin binds in the same location as
the benzyl ring of TIB. Although the planes of the aromatic rings are
inclined at about 20° to each other, they make many of the same
interactions with the walls of this part of the pocket, and both drugs
can also from specific hydrogen bonds with His-242. The presence of
bulky iodine atoms projecting from the TIB benzyl group make it too
wide to fit into the sub-pocket that accommodates the benzyl ring of warfarin.
Effect of Fatty Acids on Warfarin Binding--
Our structural
results offer a plausible explanation for the observation that binding
of fatty acids to HSA can enhance the affinity of the protein for
warfarin and some other site I ligands (22, 23, 34). The presence of up
to 3 mol of long chain fatty acids per mol of HSA can increase the
affinity for warfarin by almost a factor of three (22, 23). Fatty acid
binding to HSA induces a substantial conformational change in the
protein involving rotations of domains I and III relative to domain II (11, 12, 36). One of the principal driving forces behind this
conformational change is binding of a fatty acid molecule to the
binding site (FA2) that spans the interface between subdomains IA and
IIA and lies close to the warfarin binding site. In the absence of
fatty acid the side chain of Tyr-150 from the linker connecting helices
h2 and h3 of subdomain IB projects into the warfarin binding site (Fig.
3D). However, occupation of site FA2 by myristate displaces
domain IB, rotating helices h2 and h3 and pulling the side chain of
Tyr-150 out of the drug pocket to allow it to hydrogen-bond with the
carboxylate group of the fatty acid (FA2). In concert with the movement
of Tyr-150 the side chain of Arg-257 rotates to hydrogen-bond to the
same fatty acid carboxylate. The changes induced by fatty acid binding,
removal of a hydroxyl group from the drug pocket and repositioning of
the aliphatic portion of Arg-257, thus render the binding environment
for the coumarin moiety more hydrophobic. This appears to be the most likely explanation for the increased binding affinity of HSA for warfarin. Curiously, the binding of phenprocoumon is only marginally (~20%) enhanced under similar conditions (22). This suggests that
minor conformational changes induced by fatty acid binding in the
vicinity of the mouth of the pocket may also affect drug binding.
The enhancement of warfarin binding by fatty acids is maximal with 3-4
mol of fatty acid bound per mol of HSA (22, 23). At higher fatty acid
concentrations the affinity for warfarin falls off, an observation that
is probably attributable to direct competition between the drug and
fatty acid for binding to the pocket in IIA. This interpretation
is supported by the observation that fatty acid binds to this site
(designated FA7) in a manner that is suggestive of a low
affinity interaction (primarily because of the absence of specific
interactions between the protein and the fatty acid carboxylate group)
(12). Thus we would only expect to find displacement of the drug by
high levels of fatty acid.
Effect of pH on Warfarin Binding--
Over the pH range 6-9 HSA
undergoes a transition from the neutral to the basic form that is
linked to an increase in the affinity of the protein for a number of
site I ligands, including warfarin (which binds about 3-fold more
tightly at alkaline pH) (24). The precise nature of the structural
changes associated with the neutral-basic transition is incompletely
characterized. The transition appears largely to involve changes in
domains I and II, although there is some involvement of domain III
(32). It remains unclear whether there is any link between the
structural changes induced by shifts in pH and those induced by fatty
acid binding, although the observation that fatty acid binding lowers
the pH of the midpoint of the neutral-basic transition suggests a
possible parallel between the two effects (25). In view of the paucity
of data on this point, attempts to explain the pH effect on warfarin
binding to HSA from the crystal structure are problematic. Our results
show that the only group in the binding site that is likely to be
titratable in the range pH 6-9 is the side chain of His-242,
which makes a hydrogen bond to the drug. However, raising the pH would
deprotonate the histidine, which would be expected to reduce the
strength of its interaction with warfarin, contrary to the results of
binding experiments. It thus appears that other effects of pH upon the protein are responsible for tighter binding at alkaline pH, and further
work will be required to determine what these are.
Conclusions--
Recently, methods to study structural aspects of
drug binding using engineered fragments of HSA have been developed in
an effort to make the analysis more tractable (35, 37). Although such
approaches are likely to be very valuable, particularly because they
use fragments small enough to be analyzed structurally by nuclear
magnetic resonance techniques, truncations of the protein may give rise
to inadvertent structural perturbations of the drug binding sites. This
study and an earlier structural analysis of HSA complexed with the
general anesthetics propofol and halothane (15) demonstrate the
feasibility of high resolution crystallographic studies of the binding
of drugs to the intact protein and show that the structural information
obtained provides a richly detailed view of the binding site with which
to interpret the biochemical and biophysical data already accumulated
on drug interactions with the protein. Further work on other site I and
site II drugs will allow us to build up a much more complete picture of
drug interactions with HSA and will provide a structural basis for a
more rational approach to drug design either to exploit or avoid the
impact of HSA on drug delivery.
) enantiomers of warfarin, a widely used
anticoagulant that binds to the protein with high affinity. The
structures confirm that warfarin binds to drug site I (in subdomain
IIA) in the presence of fatty acids and reveal the molecular details of
the protein-drug interaction. The two enantiomers of warfarin adopt
very similar conformations when bound to the protein and make many of
the same specific contacts with amino acid side chains at the binding
site, thus accounting for the relative lack of stereospecificity of the
HSA-warfarin interaction. The conformation of the warfarin binding
pocket is significantly altered upon binding of fatty acids, and this
can explain the observed enhancement of warfarin binding to HSA at low
levels of fatty acid.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical domains (I-III); each domain can be
divided into subdomains A and B, which contain six and four
-helices, respectively (3, 10). Structural studies have mapped the
locations of the fatty acid binding sites (11, 12) and the primary drug
binding sites on the protein (10). The fatty acid binding sites are
distributed throughout the protein and involve all six subdomains; by
contrast many drugs bind to one of the two primary binding sites on the
protein, known as Sudlow's sites I and II (13). Although examples of
drugs binding elsewhere on the protein have been documented (13-15),
most work has focused on the primary drug sites. These investigations
have largely employed competition binding methods to investigate the selectivity of the primary drug binding sites. Drug site I, where warfarin binds, has been characterized as a conformationally adaptable region with up to three subcompartments (16-18).
-acetonylbenzyl)-4-hydroxycoumarin) binds to drug site I with
high affinity (Kd
3 µM) (13, 20,
21). A crystallographic study in 1992 confirmed subdomain IIA as the binding locus for a single molecule of warfarin but was unable to give
specific details on the binding interactions because of the limited
resolution of the data (10). Warfarin shares this binding site with a
range of other drugs (including phenylbutazone, tolbutamide, and
indomethacin) and thus competes with them for binding to HSA (13, 16,
17). Other studies have shown that low levels of fatty acids (22, 23)
or elevated pH (24, 25) may enhance the affinity of HSA for warfarin by
up to 3-fold, but the molecular mechanisms of these effects are not
well understood. To elucidate the molecular details of the
interaction of warfarin with HSA, we report here the determination of
the crystal structures of HSA-myristate complexed with both the
R-(+) and S-(
) enantiomers of the drug.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) enantiomers using established protocols (26). Crystals of HSA-myristate were harvested into solutions containing 37% (w/v) polyethylene glycol 3350, 50 mM potassium phosphate, pH 7.0. Warfarin was dissolved at
20 mM in harvest buffer at pH 12.0 with continuous stirring
for 24 h; the pH was then restored to 7.0 with concentrated HCl.
Crystals were soaked first in harvest buffer containing 10 mM R-(+) or S-(
) warfarin
enantiomer for 15 h and then in buffer containing the same
enantiomer (20 mM) for at least 24 h.
Fc and 3Fo
2Fc maps. These maps were used to guide the
positioning of the fatty acid and warfarin ligands and bound waters and
to make manual adjustments to the protein prior to further cycles of
refinement. Details of the refinement statistics are summarized in
Table I. Coordinates have been deposited with the Protein Data Bank
(identification codes are given in Table I). Figures depicting the
structure were prepared using BOBSCRIPT (28) and RASTER3D (29).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)
enantiomers (Fig. 1) up to a final concentration of 20 mM (see "Experimental Procedures").
The soaked crystals were isomorphous to crystals of HSA-myristate, and
the structures were solved by molecular replacement. Difference
electron density maps showed clear density for a single warfarin
molecule in subdomain IIA in each case (Fig.
2). The density was consistent with the
open form of the warfarin molecule (31); features corresponding to the
coumarin, benzyl, and acetonyl moieties of the drug were evident and
gave a clear indication of the overall orientation of the drug in the
pocket. For both R-(+) and S-(
) warfarin there nevertheless seemed to be a possible ambiguity in the orientation of
the coumarin moiety. However, although the density appeared to allow
the possibility of flipping the coumarin group by 180° about the
C2-C13 bond (Figs. 1 and 2), attempts to refine the drug in the
alternative orientation resulted in higher B-factors for the ligand, a
poorer fit to 3Fo
2Fc electron density maps, and significantly more residual difference density (data
not shown). The density is thus consistent with a single dominant
conformation of the bound drug for each of the enantiomers. Models for
R-(+) and S-(
) warfarin bound to HSA-myristate
were refined to 2.5 Å and have Rfree values of
25.4 and 24.8%, respectively, and good stereochemistry (Table
I).
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Fig. 1.
Schematic structures of warfarin,
phenprocoumon, and acenocoumarin. Selected positions in warfarin
that are referred to in the text are numbered in
parentheses.
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Fig. 2.
Experimental Fo Fc difference electron density map
for (A) R-(+) and
(B) S-(
) warfarin in subdomain IIA,
where Fo values are the observed
HSA-myristate-warfarin amplitudes, and Fc
values are the calculated amplitudes from the protein model from the
HSA-myristate structure (11); the phases were derived from the same
model. Oxygen atoms in the warfarin molecule are shown in
dark gray.
Data collection and model refinement statistics
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Fig. 3.
Structural details of the interaction
of the R-(+) enantiomer of warfarin with
HSA-myristate. A, overview of the
HSA-myristate-warfarin complex showing the drug binding to a single
locus in subdomain IIA. The protein secondary structure is shown
schematically, and the domains are color-coded as follows: I,
red; II, green; III, blue. The A and B
subdomains are depicted in dark and light shades,
respectively. Bound fatty acids are shown in a space-filling
representation and colored by atom type (carbon, gray;
oxygen, red). Where two fatty acid molecules bind in close
proximity, one of them is shown in a darker shade of gray.
Warfarin is depicted in the same space-filling representation, with
yellow carbon atoms. B, side view of warfarin
pocket showing a cutaway view of the surface of the binding pocket. The
surface is colored to indicate electrostatic potential, with
blue representing basic patches. The view is rotated by
180° about a vertical axis relative to the view shown in
A. The figure was prepared using GRASP (38). C,
stereo view of the warfarin binding pocket showing the amino acids
lining the pocket. The view is rotated by 90° relative to the view in
A. Side chain atoms are colored by atom type (carbon,
gray; nitrogen, blue; oxygen, red);
water molecules bound adjacent to warfarin are depicted as cyan
spheres. D, impact of conformational changes induced by
fatty acid binding on drug site I and potential implications for
warfarin binding. The structures of defatted HSA and
HSA-myristate-warfarin were superposed by aligning the positions of
residues belonging to domain II (197). The figure shows details of
warfarin binding to HSA-myristate; superposed on this and shown in
semitransparent mode are helices h2 and h3 of subdomain IB.
) enantiomers bind in the
pocket in almost identical conformations, both in the open
configuration (31). The coumarin and benzyl moieties of the
R-(+) and S-(
) forms are nearly perfectly
superimposable, presumably because stabilization of the open
conformation allows these groups to rotate about the C2-C13 bond. The
main difference in the drug conformations occurs in the acetonyl group,
which branches from the chiral carbon and is located at the mouth of the pocket. However, at 2.5 Å this difference is barely detectable in
our electron density maps. The finding that the enantiomers bind
in essentially the same way to HSA is consistent with the observation
that they have similar binding affinities for the protein. At 25 °C,
close to the temperature of our diffraction data collection, the
R-(+) and S-(
) enantiomers have dissociation constants of 3.8 and 2.9 µM, respectively (21), although
the difference is slightly more pronounced at lower temperatures (20, 21).
of Arg-222. Closer to the pocket
entrance the O3 of the acetonyl group lies 3.3 Å from NH2 of
Arg-222. The combination of electrostatic interactions made by O2, O3,
and O4 probably helps to fix the orientation of the bound warfarin, but
complementarity between the shape of the drug and the pocket is clearly
also important for binding. There is no significant difference in the
electrostatic contacts made by the two enantiomers.
Potential electrostatic interactions between warfarin and HSA
Van der Waals interactions between warfarin and HSA
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Delta Biotechnology Ltd. for purified recombinant HSA and the staff at DESY Hamburg (Germany) for help with data collection. We are very grateful to Peter Brick for critical reading of the manuscript.
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FOOTNOTES |
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* This work was funded by grant support from the Biotechnological and Biological Sciences Research Council, UK.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1h9z and 1ha2) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Recipient of a Ph.D. studentship from the Medical Research
Council, UK.
§ Present address: The Burnham Inst., 10901 N. Torrey Pines Rd., La Jolla, CA 92037.
To whom correspondence should be addressed. Tel.:
44-20-7594-7632; Fax: 44-20-7589-0191; E-mail: s.curry@ic.ac.uk.
Published, JBC Papers in Press, April 2, 2001, DOI 10.1074/jbc.M100575200
2 A. Leslie, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: HSA, human serum albumin; TIB, 2,3,5-triiodobenzoic acid.
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