From the Department of Chemistry, University of
Edinburgh, EH9 3JJ United Kingdom, the ¶ Department of
Biochemistry, College of Medicine, University of Vermont, Burlington,
Vermont 05405, and the § Department of Chemistry,
University of
Hong Kong, Hong Kong, Peoples Republic of China
Received for publication, June 2, 2000, and in revised form, October 11, 2000
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ABSTRACT |
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Several bismuth compounds are currently used as
antiulcer drugs, but their mechanism of action is not well established.
Proteins are thought to be target sites. In this work we establish that the competitive binding of Bi3+ to the blood serum
proteins albumin and transferrin, as isolated proteins and in blood
plasma, can be monitored via observation of 1H and
13C NMR resonances of isotopically labeled
[ Bismuth compounds have long been associated with medicine for the
treatment of a variety of gastrointestinal disorders including diarrhea, constipation, gastritis, and ulcers (1-4). The effectiveness of bismuth has been attributed to its bactericidal action against the
Gram-negative bacterium, Helicobacter pylori. There is also a growing interest in using compounds containing radioactive bismuth isotopes as targeted radiotherapeutic agents (5). However, the
molecular basis for the mechanism of action of bismuth drugs is not
well understood, including bismuth-induced toxicity, especially encephalopathy, which led to the withdrawal of bismuth drugs in France
and Australia in the 1970s (1). The diagnosis of encephalopathy is
generally defined by the detection of bismuth in blood, plasma or
serum, the so-called "Hillemand safety level" (6, 7). Bismuth is
primarily present in red blood cells, possibly binding to glutathione,
with the remainder in serum or plasma (8-10). The speciation of
bismuth in blood plasma, and in particular the nature of interactions
of Bi3+ with plasma proteins, are in need of investigation.
Recently we have found that the binding of Bi3+ to human
serum transferrin (hTF)1 and
recombinant N-lobe of transferrin is unexpectedly strong (11, 12).
Transferrin is a single-chain glycoprotein (80 kDa) present in blood at
a concentration of about 35 µM, and consists of two
similar lobes, each of 40 kDa, connected by a short peptide. Its normal
function in blood is to carry iron between sites of uptake,
utilization, and storage (13-16). It contains two specific iron-binding sites per molecule, one in the N-terminal lobe and one in
the C-terminal lobe. Iron binds as Fe3+ in a cleft formed
by two domains in each lobe. Iron cannot bind strongly without
concomitant binding of a synergistic anion. Since transferrin is only
about 30% saturated with iron in normal serum (13, 17, 18), there is
potential binding capacity for other metal ions that enter the blood.
This has led to the idea that transferrin acts as a "delivery
system" for therapeutic, diagnostic or toxic ions, including
Ga3+, Ru3+, and Al3+ (19-21).
Recently we have shown that Bi2-hTF can block both membrane binding and cellular uptake of 59Fe-hTF into BeWo placental
cancer cells (22). It is therefore now important to establish whether
Bi3+ binding to transferrin can occur under physiologically
relevant conditions, especially in the presence of excess albumin and
in blood plasma itself. We have shown previously that the order of lobe
loading of hTF with metal ions can readily be determined via
two-dimensional 1H,13C NMR
studies of recombinant [ Previous investigations of the interaction of Bi3+ with
serum albumin has led to the suggestion that albumin may be the major target for Bi3+ in plasma (8), especially since albumin has
a free thiolate group at Cys34. Human serum albumin, the
most abundant protein in blood at a concentration of about 40 mg
ml In the present work, the binding of a bismuth antiulcer drug to human
serum transferrin in aqueous solution in the presence of a large excess
of albumin and to recombinant transferrin (N-lobe of transferrin and
the mutant I132M of N-lobe of transferrin labeled with
[ Materials--
Recombinant N-lobe hTF/2N (residues 1-337) was
expressed in baby hamster kidney cells using a pNUT plasmid with
L-[
A 50 mM stock solution of [Bi(cit)] Preparation of NMR Samples--
I132M [ NMR Spectroscopy--
NMR spectra were recorded on a Bruker
DMX500 spectrometer at 310 K. For one-dimensional 1H NMR
spectra, 400 to 1200 transients were acquired with 6-µs (50o) pulses and 16,384 data points during the 2-s pulse
delay and the water resonance was suppressed via presaturation. For
two-dimensional 1H,13C heteronuclear single
quantum coherence spectra (HSQC) experiments, the sequence was
optimized for 1J (1H-13C) = 140 Hz, and 16 to 32 transients were acquired using 2,048 data points
in the f2 dimension (1H), 32 to 64 increments
in the f1 dimension, 13C frequency width of 3 kHz, and relaxation delay of 1.6 s. The 13C spins were
decoupled using the GARP sequence (31). After zero-filling to
4,096 × 1,024 data points, unshifted Gaussian functions were used
for processing in both dimensions. Water suppression was achieved by a
combination of presaturation and pulsed-field gradients. One-dimensional HSQC NMR spectra (or 13C-edited
1H NMR spectra) were recorded using the first increment of
the two-dimensional HSQC sequence. Resolution enhancement was achieved using a combination of exponential (1.5 to 10 Hz line-broadening) and
unshifted sine-bell functions (32). Peaks were referenced to sodium
3-(trimethylsilyl)propionate-2,2,3,3-d4
via the external Determination of Bi--
HSA (concentration about 0.1 mM) was incubated with different mole ratios of RBC
in 0.1 M Tris-HCl buffer, pH 7.4, overnight at 310 K. Unbound RBC was separated via gel filtration using a Superose 12 column
and a FPLC system (Amersham Pharmacia Biotech). The concentration of
albumin samples was about 6 mg/ml and 500 µl of the protein solution
was loaded onto the column. Elution conditions were 0.1 M
Tris-HCl, pH 7.4, and flow rate 0.5 ml/min. The fractions eluting from
10 to 14 ml were collected and the bismuth content was determined using
a CETAC Microneb 2000 direct injection nebulizer (CETAC Technologies,
Omaha, NE) coupled with a VG PlasmaQuad PQ2 ICP-MS instrument (VG
Elemental, Winsford, Cheshire, UK). Details of the optimization
procedure for the DIN-ICP-MS system and measurement conditions for
bismuth have been described previously (33).
Determination of Albumin Thiol Content--
The free SH content
of albumin was determined after incubation with RBC or
[Bi(cit)] NMR Studies of Bi3+ Binding to I132M hTF/2N and
Comparison with Fe3+
One-dimensional 1H NMR--
It has been shown
previously that 1H NMR spectra of human serum
transferrin are complicated by the overlap of the very large number of
resonances present and by their broadening due to the slow tumbling of
this 80-kDa protein (36). However, the high-field region of the
spectrum of the N-lobe is relatively well resolved. High field-shifted
resonances have been assigned to protons from residues around
Trp128, i.e. Leu122, and
Ile132 (37, 38). The mutation of Ile132 to Met
should lead to the disappearance of the resonance at
For comparison, titrations of Fe3+ (added as
Fe(NTA)2) with the mutant protein were also performed under
similar conditions (pH* 7.8, 310 K and 10 mM bicarbonate)
and the results are shown in Fig. 1B. Upon addition of 0.5 mol eq of Fe3+, the resonance at Two-dimensional 1H,13C HSQC NMR--
The
two-dimensional 1H,13C HSQC NMR spectrum of
I132M hTF/2N and after addition of 0.5 mol eq of Bi3+ or
Fe3+, in the presence of 10 mM bicarbonate, are
compared in Fig. 2. As expected, six
cross-peaks were observed for the apo-protein, five of which have
similar chemical shifts as those observed for the wild-type protein.
These have been assigned previously on the basis of single-site
mutations combined with other considerations (39, 40). Therefore the
sixth peak, at 1.51/17.98 ppm, can be assigned to Met132.
On addition of 0.5 mol eq of Bi3+ (added as either RBC or
as [Bi(NTA)]), a notable decrease in intensity of the latter peak
occurred and a new peak appeared at 1.45/17.93 ppm, which can be
associated with the bound form of the protein. Other new peaks, which
can be assigned to Met109 and Met309, also
appeared and shifted slightly in both 1H and
13C dimensions (Table I).
Further addition of Bi3+ (1.0 mol eq) caused the
disappearance of the peaks at 1.51/17.98, 1.94/16.15, and 2.15/16.16
ppm and led to a further increase in the intensity of the new peaks.
These changes were observed more clearly in high-resolution
one-dimensional 13C-edited 1H spectra (see
below, Fig. 3).
Addition of 0.5 mol eq of Fe3+ (added as
[Fe(NTA)]2) to I132M hTF/2N caused similar changes in the
two-dimensional HSQC spectrum as for 0.5 mol eq Bi3+,
except that the peak for Met132 for the bound form was
significantly broadened. The resonance for Met132 in the
apo-protein disappeared after addition of 1.0 mol eq of Fe3+. The changes to the shifts of the other Met resonances
were identical to those observed on addition of 1.0 mol eq of
Bi3+.
Binding of Bi3+ to I132M hTF/2N in the Presence of
Excess of Albumin--
The 1H and 13C NMR
chemical shift changes induced by metal ions (e.g.
Fe3+ and Bi3+) provide convenient probes for
investigation of Bi3+ translocation between transferrin and
proteins such as albumin. These experiments were performed using low
concentrations of I132M hTF/2N (150 µM) in the presence
of 12 mol eq of HSA or rHA (1.8 mM), pH* 7.8, 10 mM bicarbonate, 310 K. We choose an
[albumin]/[transferrin] ratio of 12:1 to mimic biological
conditions. The concentration of albumin in blood plasma (about 0.63 mM) is about 18 times higher than that of transferrin
(about 35 µM), but hTF is only about 30% saturated with
Fe3+. The 1H,13C two-dimensional
HSQC NMR spectrum of this protein mixture shows sharp resonances from
the six labeled Met residues of transferrin and broadened (natural
abundance) resonances from albumin. The Met132 peak was
overlapped with peaks from albumin (data not shown). Since I132M hTF/2N
is present at low concentration, one-dimensional 13C-edited
1H NMR spectra were recorded over a period of 30 min each.
Fig. 3 shows the 13C-edited 1H NMR spectrum of
I132M hTF/2N in the absence and presence of recombinant albumin (rHA).
Broad resonances at 1.70 and from 1.45 to 1.10 ppm from albumin were
filtered out with resolution enhancement using a combination of
unshifted sine-bell and exponential functions prior to Fourier transformation.
The antiulcer compound, ranitidine bismuth citrate, was added in 0.25 mol eq steps to I132M hTF/2N in the presence of 12 mol eq of rHA, 10 mM bicarbonate. With increase in Bi3+
concentration, a new peak at 1.45 ppm appeared. This can be assigned to
Met132 in Bi-I132M hTF/2N, and it gradually increased in
intensity, reaching a maximum after 1 mol eq of Bi3+ had
been added. The peak for Met132 in the apo-protein
simultaneously decreased in intensity and finally disappeared (Fig. 3).
The change in the peak for Met309 (2.15 ppm) was observable
more clearly in one-dimensional 1H{13C}
spectra: it gradually decreased in intensity with the addition of
Bi3+ and finally disappeared. The resonance for
Met309 is probably that at 2.14 ppm, which was overlapped
with Met256, as can be judged from the increased intensity
in the two-dimensional HSQC spectrum (data not shown). The peak for
Met109 also appears to shift to low field by 0.03 ppm. The
same chemical shift changes were observed after addition of
Bi3+ to I132M hTF/2N in the presence of 12 mol eq of HSA.
The lack of effect of albumin on Bi3+ binding to this
transferrin N-lobe is clearly illustrated in Fig. 3C, which
shows the integrated intensity of the peak for Met132 after
addition of various amounts of Bi3+ in the absence and
presence of albumin.
Since the concentration of albumin present in the sample is much higher
than that of transferrin, the 1H NMR spectrum is dominated
by peaks from albumin. The aromatic region of spectra of these mixed
protein solutions (hTF/2N + 12rHA or + 12HSA) was almost identical with
and without addition of Bi3+, particularly the resonance at
7.632 ppm, which has been previously assigned (41, 42) to
His3 of albumin (data not shown).
Uptake of Bi3+ by Transferrin in Plasma
The concentration of transferrin in human plasma is about 35 µM. It is only about 30% saturated with iron (18)
and therefore has about 50 µM capacity for binding to
other metal ions. To determine if transferrin is a target for bismuth,
isotopically labeled [ Even with resolution enhancement, the peak for Met132 was
still overlapped with other peaks in the 13C-edited
1H NMR spectrum (data not shown). Therefore only the
two-dimensional HSQC method was used. The two-dimensional HSQC spectrum
of this solution containing 100 mM KCl and 20 mM bicarbonate is shown in Fig.
4. Surprisingly, the peak for
Met109 became severely broadened but the rest of the Met
cross-peaks from transferrin were clearly observed. Many other
cross-peaks are present but are difficult to assign, partly due to the
limited frequency width used (12 ppm in 13C dimension). The
peaks at about 1.46/17.2 ppm, and 1.67/15.8 ppm (folded in
13C dimension) can be assigned to Ala and Lys residues,
respectively, of albumin, and the peaks at about 1.24/19.2 ppm to
lipids in plasma. After addition of 0.5 mol eq of RBC (relative to the
available transferrin-binding sites), the peak for Met132
(1.51/17.98 ppm) in apo-hTF/2N decreased in intensity and the peak at
1.45/17.93 ppm for Bi3+-I132M hTF/2N increased in
intensity. Similarly, the peak for Met109 (1.94/16.15 ppm)
disappeared and a new peak (bound form) appeared at slightly lower
field, and that for Met309 (2.15/16.16 ppm) shifted to high
field. The cross-peak for Met132 in the apo-protein almost
disappeared and the analogous peak for the bound-form further increased
in intensity (Fig. 4). After addition of 1.0 mol eq of RBC, this
behavior was similar to that observed for I132M hTF/2N with and without
12 mol eq of serum albumin or recombinant albumin under same
conditions. Interestingly, with Bi3+ bound to the protein,
the peak for Met26 became sharper and observable.
-13C]Met transferrin. We show that
Met132 in the I132M recombinant N-lobe transferrin mutant
is a sensitive indicator of N-lobe metal binding. Bi3+
binds to the specific Fe3+ sites of transferrin and the
observed shifts of Met resonances suggest that Bi3+ induces
similar conformational changes in the N-lobe of transferrin in aqueous
solution and plasma. Bi3+ binding to albumin is nonspecific
and Cys34 is not a major binding site, which is surprising
because Bi3+ has a high affinity for thiolate sulfur. This
illustrates that the potential target sites for metals (in this case
Bi3+) in proteins depend not only on their presence but
also on their accessibility. Bi3+ binds to transferrin in
preference to albumin both in aqueous solution and in blood plasma.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-13C]Met-hTF (23). It is known
that the strength of binding to the two lobes is slightly different,
and that Fe3+ is primarily situated in N-lobe in serum (14,
18).
1 (about 0.63 mM, > 10 times that of
transferrin), is a single-chain 66.5-kDa protein, which is largely
-helical, and consists of three structurally homologous domains
(24). It is the major transport protein for unesterified fatty acids,
but is also capable of binding an extraordinarily diverse range of
metabolites, drugs, organic compounds, and metal ions, e.g.
Ca2+, Zn2+, Cu2+, and
Ni2+ (25, 26).
-13C]Met) in intact blood plasma has been monitored
directly under physiologically relevant conditions using
1H,13C NMR spectroscopy. The introduction of
Met132 into the N-lobe provides a convenient monitor for
metal binding since this residue occupies a similar site within helix 5 of the N-lobe and forms part of the hydrophobic patch around
Trp128
(Leu122-Trp128-Ile132) as
Met464 in the C-lobe
(Val454-Trp460-Met464). The
interaction of bismuth with human albumin was also studied. Surprisingly, we found that Bi3+ still binds to the
iron-binding sites of transferrin even in the presence of a large
excess of albumin.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-13C]methionine in the growth medium,
and purified as described previously (27, 28). A gene for the mutant
I132M protein was created by site-directed mutagenesis using previously
published methods (27, 29). Iron was removed from proteins by treatment
with a metal-removal buffer containing 1 mM NTA, 1 mM EDTA, and 0.5 M sodium acetate, pH 4.9, using ultrafiltration Centricon 10 ultrafilters (Amicon). Human serum
albumin (HSA) was purchased from Sigma as essentially globulin-free and
fatty acid-free and was purified via ultrafiltration (Centricon 10)
using 0.1 M KCl and washing 3 times (each 1 h). It was
then lyophilized. Recombinant human albumin (rHA) was supplied by Delta
Biotechnology Ltd. (batches GA 950202 and R970103). Samples of rHA were
dialyzed against 100 mM ammonium bicarbonate, pH 7.9, and
freeze-dried. Ranitidine bismuth citrate (RBC) and bismuth citrate
[Bi(Hcit)] were provided by GlaxoWellcome plc. NaHCO3,
KCl, 5,5'-dithiobis(2-nitrobenzoic acid), and other chemicals were
purchased either from Aldrich or Sigma with the highest quality and
used as received. Crystalline [Bi(NTA)] was synthesized according to
a literature procedure (30), and had a satisfactory elemental analysis.
was
prepared by addition the minimum amount of ammonia solution to a
suspension of [Bi(Hcit)] until the solution became clear. The final
pH of this solution was about 7, and it was then diluted before use. A
solution of Fe(NTA)2 was prepared from an iron atomic
absorption standard (1000 ppm in 1% HNO3, Aldrich) and 2 mol eq of H3NTA (Aldrich), followed by pH adjustment to
between 6.0 and 7.0. This was lyophilized and redissolved in
D2O before use. A [Bi(NTA)] solution was prepared by
dissolving a known amount of [Bi(NTA)] in D2O.
-13C]Met
hTF/2N (0.15 mM) and mixtures with either rHA (1.8 mM) or HSA (1.8 mM) were prepared in
D2O containing 0.1 M KCl. Prior to
Bi3+ or Fe3+ titrations, an aliquot of
concentrated NaHCO3 (0.25 M) was added to the
samples to give a final concentration of 10 mM, and pH* values were adjusted using NaOD or DCl. For the intact blood plasma experiments, blood from a male healthy volunteer was collected by
venipuncture into lithium-heparinized vacutainers, and the plasma was
separated by centrifugation at 6000 rmp for 20 min at 277 K, and stored
frozen until used for NMR measurements. I132M hTF/2N was added to 1.2 ml of intact blood plasma to give a hTF/2N concentration of 50 µM and the concentration of this sample was doubled by
freeze-drying and reconstitution in 0.6 ml of D2O, followed
by addition of concentrated NaHCO3 to a final concentration of 20 mM. The pH* was then measured. After addition of
Bi3+, samples were left to equilibrate for at least 30 min
at 310 K. All experiments on reconstituted blood plasma were carried out at pH* 7.8 since it was possible to maintain this as a stable pH*
value during the course of the long NMR data accumulations. If the
initial pH* value was lower, it tended to drift upwards during the
experiment. The 1H,13C chemical shifts of the
[
-13C]Met resonances of transferrin were insensitive
to pH over the range of 7-8.8, and NMR experiments on Bi3+
loading of transferrin with [Bi(NTA)] and ranitidine bismuth citrate
gave the same results at pH* 7.4 and pH* 7.8.
-CH3 peak of L-methionine
(15.14 ppm) for 13C and via formate (8.465 ppm, a minor
impurity always present in the protein) for 1H.
using Ellman's reagent,
5,5'-dithiobis(2-nitrobenzoic acid). HSA or rHA were incubated with
different mole ratios of RBC or [Bi(cit)]
overnight
(>12 h) in 0.1 M Tris-HCl buffer, 310 K, pH 7.4, and unbound bismuth was removed by ultrafiltration using Centricon 10 ultrafilters. The concentrations of HSA and rHA were determined using
279 values of 35,300 and 37,100 M
1 cm
1, respectively (25, 34),
and the amount of nitrobenzoic acid thiol generated was calculated
using
412 = 13,600 M
1
cm
1 (35).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
0.603 ppm, which
has been previously assigned as
CH2 of
Ile132. Indeed, this was found to be the case (Fig.
1). Other changes were also observed in
the spectrum of the mutant in comparison to that of wild-type hTF/2N.
For example, the resonance at
0.170 ppm for hTF/2N disappeared, and
the peak for
CH3 of Leu122 (
0.339 ppm in
hTF/2N) shifted to
0.324 ppm. Addition of 0.5 mol eq of
Bi3+ (as [Bi(NTA)]) caused new peaks to appear at
0.160
ppm (C'), and
0.255 ppm (B'), and further addition of
Bi3+ (total 1.0 mol eq) increased the intensity of both C'
and B' but decreased that of peak B significantly (Fig.
1A).
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Fig. 1.
The high-field region of the 1H
NMR spectrum of apo-I132M hTF/2N and its Bi3+ and
Fe3+ complexes in the presence of 10 mM
bicarbonate 0.1 M KCl, pH* 7.8. A, apo-I132M hTF/2N (bottom), and after addition
of 0.5 (middle), and 1.0 mol eq of [Bi(NTA)];
B, apo-I132M hTF/2N (bottom), and after addition
of 0.5 (middle) and 0.9 mol eq of [Fe(NTA)2];
C, stack plot showing the changes in shifts of the methyl
protons of Leu122 for wild-type hTF/2N and I132M mutant
after metal binding.
0.324 ppm decreased in
intensity, and almost completely disappeared with 0.9 mol eq
Fe3+ present. Broad new peaks at
0.004 and
0.392 ppm
appeared and increased in intensity. It is reasonable to assume from
these titration studies that the resonance at
0.324 ppm consists of two overlapped peaks, one of which (peak B) can be assigned to the
CH3 of Leu122 as judged from the change in
pattern of this peak on titration of both wild-type and mutant proteins
with metal ions. The other peak (C) cannot be assigned. Two-dimensional
total correlation spectroscopy and NOESY experiments support these
assignments (data not shown). Peak B has identical associated NOESY
cross-peaks for both wild-type and mutant hTF/2N, which suggests that
peak B belongs to the
CH3 of Leu122. Similar
cross-peak patterns were observed for B and B' in the NOESY spectrum,
which indicates that peak B' for the metal-bound protein is the
analogue of peak B. A comparison of the changes in the chemical shifts
of the
CH3 peak of Leu122 after binding of
hTF/2N and I132M-hTF/2N to Bi3+ and Fe3+ is
shown in Fig. 1C.
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Fig. 2.
Two-dimensional
1H,13C HSQC NMR spectra of
[ -13C]Met I132M hTF/2N in 10 mM bicarbonate, 0.1 M KCl at pH*
7.8. A, apo-hTF/2N; B, after addition
of 0.5 mol eq of ranitidine bismuth citrate; and C,
apo-hTF/2N after addition of 0.5 mol eq of Fe3+ (added as
[Fe(NTA)2]).
1H and 13C NMR chemical shifts of the
[-13C]Met resonances of apo-I132M hTf/2N (in 10 mM HCO
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Fig. 3.
Effect of the antiulcer compound ranitidine
bismuth citrate on the one-dimensional 13C-edited
1H NMR spectrum of apo-I132M hTF/2N. A, in
the absence; and B, in the presence of 12 mol eq of
recombinant serum albumin. C, variation in intensity of
the13C-edited 1H NMR peak for
Met132 in [ -13C]Met-I132M hTF/2N with
increase in the Bi3+/protein ratio. Open
symbols, apo-hTF/2N; filled symbols, Bi-hTF/2N;
circle, hTF/2N; square, hTF/2N + 12 mol eq of
rHA; triangle, hTF/2N + 12 mol eq of HSA.
-13C]Met I132M hTF/2N (50 µM) was directly added to human plasma. The whole plasma
concentration (including the added transferrin) was lyophilized, and
the sample was redissolved in half-volume of the original plasma
solution. This gave an I132M hTF/2N concentration of 100 µM.
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Fig. 4.
Two-dimensional
1H,13C HSQC NMR spectra of blood plasma
containing 100 µM
[ -13C]Met I132M apo-hTF/2N.
A, before; B, after addition of Bi3+
(ranitidine bismuth citrate); and C, slices through
two-dimensional 1H,13C HSQC spectra in the
1H dimension (corresponding to the 13C signal
of
SCH3 of Met132).
The shift of the peak for Met132, which is in the
hydrophobic patch of helix 5, is similar to that observed after direct
addition of Bi3+ to I132M apo-hTF/2N (Fig. 2).
A second experiment was carried out with blood plasma containing twice the concentration of I132M (200 µM), but the behavior of the Met two-dimensional cross-peaks on titration with RBC was similar. The normal one-dimensional 1H NMR spectrum of plasma in the His region was identical in the absence and presence of Bi3+, especially the peak for His3 of albumin (7.632 ppm), which has previously been used as an indicator for drug binding at Cys34 (41).
The intact protein as [-13C]Met-transferrin was also
added directly to 1.2 ml of human plasma to give a concentration of 35 µM, and the concentration of the sample was doubled by
freeze-drying and dissolving in 0.6 ml of D2O in the
presence of 20 mM sodium bicarbonate. Most of the Met
resonances were observable in the two-dimensional HSQC spectrum except
those for Met26, Met309, and Met389
(Fig. 5). Other peaks from plasma were
also observed from groups present at relatively high concentrations
such as Lys (from albumin) and lipids. The notable change was for
Met464 in the C-lobe from its apo-position (1.38/16.30 ppm
for 1H/13C) to Bi3+ bound form
(1.18/18.2 ppm for 1H/13C) after addition of 1 mol eq of ranitidine bismuth citrate. A similar change also occurred
for Met499 in the C-lobe.
|
Interactions of Bismuth Complexes with Albumin
Effect of Bismuth on the Free Thiol Content of Albumin--
The
free thiol of albumin at Cys34 is a potentially strong
binding site for Bi3+. The thiol contents of human serum
albumin and recombinant human albumin were determined before and after
reaction with bismuth citrate (either RBC or [Bi(cit)])
by the 5,5'-dithiobis(2-nitrobenzoic acid) method. The rHA
(recombinant) sample contained 0.77 ± 0.01 mol of thiol/mol of
protein, while thiol content of (isolated) HSA was significantly lower,
only 0.29 ± 0.01 mol/mol HSA. After reaction with various amounts
of bismuth citrate in 0.1 M Tris-HCl buffer at pH 7.4 for
12 h, the SH contents decreased by less than 12%, from 0.77 to
0.68 for rHA and from 0.29 to 0.26 for HSA, respectively. This suggests that little Bi3+ binds to Cys34 of albumin.
Determination of Amount of Bismuth Bound to Human Serum
Albumin--
Various mole ratios of ranitidine bismuth citrate were
reacted with albumin in 0.1 M Tris-HCl buffer at pH 7.4 and
equilibrated overnight at 310 K. Albumin-bound bismuth was then
separated from free bismuth by gel filtration chromatography. The
Bi3+ content of the albumin fractions was measured by
DIN-ICP-MS (data not shown). The amount of Bi3+ bound to
albumin increased almost linearly with increase in added RBC and did
not reach saturation even with 25 mol eq of RBC present. The gel
filtration chromatograms of control albumin and its complex with
bismuth were very similar both in terms of peak intensity and retention
time (data not shown) suggesting that bismuth does not cause
aggregation of the protein. When 40 mol eq of glutathione (relative to
the measured Bi3+) was added to the albumin fraction, a new
broad band centred at about 350 nm gradually increased in intensity in
a multiphase process, and reached a maximum intensity over a period of
3 h (Fig. 6). This is in contrast to
the reaction of bismuth citrate alone with 40 mol eq of glutathione
under similar conditions which was complete within minutes. The band at
350 nm is a typical Bi-S absorbance indicating formation of
[Bi(SG)3] (9).
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DISCUSSION |
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Bismuth compounds are widely used as antiulcer drugs and recently
we have shown (22) that bismuth transferrin, Bi2-hTF, exhibits marked dose-dependent effects on membrane binding
and cell uptake of 59Fe-hTF by placental BeWo cells. This
suggested that bismuth transferrin is recognized by the transferrin
receptor. The present study was undertaken to determine whether
Bi3+ can bind to transferrin under physiological
conditions, especially in the presence of excess albumin, and in blood
plasma itself. Previously we have shown that NMR can be used to monitor
the uptake of metals into the individual lobes of transferrin (23). The 1H,13C NMR cross-peak for Met464 of
human transferrin is a sensitive indicator of metal binding to the
C-lobe since significant chemical shift changes are induced in both
1H and 13C dimensions. In the N-lobe of intact
hTF, however, there is lack of this kind of sensitive indicator.
Met464 is situated in the hydrophobic patch
(Val454-Trp460-Met464) of helix 5 in the C-lobe (Fig. 7), which backs onto
the metal-binding site and H-bonds to the synergistic anion (43, 44).
In the N-lobe there is a similar hydrophobic patch in helix 5 near the metal-binding site, consisting of Leu122,
Trp128, and Ile132 (Fig. 7) (45). The analogue
of Met464 is Ile132 in human serum transferrin,
but is Met132 in cow and pig transferrin (46), which
suggests that I132M is a structurally conservative substitution. To
provide a possible sensitive indicator for metal ion binding in the
N-lobe of human serum transferrin and to investigate the similarity
between the two lobes of transferrin, Ile132 was mutated to
Met using site-directed mutagenesis. It is easy to produce N-lobe
protein in this way in the quantities required for NMR. In contrast,
recombinant C-lobe is difficult to prepare, but the N-lobe and C-lobe
metal binding constants are usually close (47).
|
The 1H NMR spectrum of I132M hTF/2N was similar to that of
wild-type hTF/2N in the both high-field and His C2H regions, except for
the disappearance of the peak for the CH2 of
Ile132 at
0.603 ppm. Both Bi3+ and
Fe3+ induce similar chemical shift changes for the high
field-shifted peak for
CH3 of Leu122 in the
mutant and wild-type hTF/2N (Fig. 1). This suggests that the overall
structure of the mutant is similar to that of wild-type hTF/2N. This
was also confirmed by molecular modeling, which showed that the protein
backbone fold of the mutant is almost identical to that of the
wild-type protein. Six of the 10 lowest energy structures placed the
side chain of Met132 above Trp128 (data not
shown), a situation which would give rise to a ring current shift for
the
-CH3 of Met132.
In the two-dimensional 1H,13C NMR spectrum of
apo-I132M hTF/2N, the -13CH3 resonance of
Met132 exhibits a significant 1H NMR high-field
shift compared with the rest of the Met peaks, as does the analogous
cross-peak for Met464 in the C-lobe. Only small changes in
shifts of the Met132 resonance (
0.06/
0.05 ppm
for 1H and 13C, respectively, Table I) occurred
when Bi3+ or Fe3+ binds to the mutant I132M
hTF/2N, in contrast to the large shifts for Met464 (
0.20/1.90 ppm for 1H and 13C, respectively)
suggesting that the structural changes in helix 5 on loading the
protein with metal ions are slightly different for the N- and C-lobes.
X-ray crystallographic studies have shown that when metals bind and
domain closure occurs, helix 5 pivots on helix 11 and that a domain
movement of about 54 ° occurs in the N-lobe but only about
15o rotation in the C-lobe (48, 49).
Our studies suggest, for the first time, that transferrin should be considered as a potential mediator for bismuth transport in blood plasma. Previously, it has been assumed (8) that albumin, the most abundant protein in blood serum with a free thiol group at Cys34, is a target site for bismuth drugs, since Bi3+ is known to have a high affinity for thiolate sulfur. Glutathione, a thiolate sulfur-containing peptide (GSH), for example, can readily displace Bi3+ from its complexes with citrate and EDTA at biological pH values (9). Recent reports (8) have shown that only 2% of albumin molecules bind to Bi3+ if binding is assumed to occur at the free thiol group of Cys34 (pKa about 5 (50)). In this work we have demonstrated that binding of Bi3+ to albumin is nonspecific; even 25 mol eq of Bi3+ did not saturate albumin, and Cys34 is not blocked by Bi3+ binding. Previous 1H NMR studies of albumin have shown that the imidazole CH resonances of His3 are sensitive to the oxidation of Cys34 and to the formation of adducts with gold antiarthritic drugs (41) probably because such reactions lead to movement of the side chain of Cys34 which is communicated to His3 via intervening helices. The His regions of 1H NMR spectra of albumin in the presence of I132M hTF/2N or of blood plasma in the presence of intact hTF were found to be almost identical after addition of bismuth compounds (data not shown) which provide further evidence that Cys34 is not a major binding site for Bi3+.
We have successfully used two-dimensional HSQC NMR spectroscopy to probe changes of Met resonances of transferrin in solution in which the concentration of albumin is 10 times higher. The observation of similar changes in the chemical shifts of the Met resides of I132M hTF/2N on binding Bi3+ in the presence or absence of a large excess of albumin, and even in blood plasma, suggests that similar conformational changes are induced by Bi3+ under these conditions. Such structural changes could be important for recognition by the transferrin receptor. Bi3+ was also observed to bind to intact transferrin in the presence of a large excess (12 mol eq) of serum albumin or recombinant albumin and a similar behavior was observed in blood plasma. Our findings may have implications for the mechanism of neurotoxicity of bismuth drugs (encephalopathy). For a long time it has not been clear how bismuth is transferred to the brain. It is generally accepted that the diagnosis of bismuth encephalopathy can be confirmed by the detection of high Bi3+ levels in whole blood, serum, or plasma, the so-called Hillemand safety level (6). It is likely that once bismuth has entered into blood it is transported by transferrin, in a similar manner to Al3+. Al3+ deposition in the brain is known to cause dialysis encephalopathy and this neurotoxicity is related to transferrin transportation and transferrin receptor recognition in the brain (51).
Selective labeling of the protein in combination with inverse NMR
detection is a powerful method for probing the structure and dynamics
of high molecular mass proteins, and provides an approach for
investigating the translocation of metallo-drugs (and other drugs)
between proteins and enzymes at concentrations of biologically
relevance without separation, and can also be applied to protein-ligand
(in this case for drug screening) (52) and protein-protein interactions.
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ACKNOWLEDGEMENTS |
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We are grateful to Professor Ross MacGillivray and Bea Tam (University of British Columbia) for providing plasmids, Drs. John A. Parkinson and John Parkinson for assistance with NMR and molecular modeling, and Dr. John Woodrow (Delta Biotechnology) for providing recombinant albumin. We also acknowledge use of the Protein Data Bank (Brookhaven National Laboratory and Structural Bioinformatics).
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FOOTNOTES |
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* This work was supported by GlaxoWellcome, the Engineering and Physical Sciences Research Council (EPSRC), Biotechnology and Biological Sciences Research Council (BBSRC), Wolfson Foundation, the University of Hong Kong, and the Committee of Vice-Chancellors and Principals (Overseas Research Student award to H. L.).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.
To whom correspondence should be addressed: Dept. of
Chemistry, University of Edinburgh, King's Buildings, West Mains Road, Edinburgh EH9 3JJ, United Kingdom Tel.: 44-131-650-4729; Fax: 44-131-650-6452; E-mail: p.j.sadler@ed.ac.uk.
Published, JBC Papers in Press, December 7, 2000, DOI 10.1074/jbc.M004779200
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ABBREVIATIONS |
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The abbreviations used are: hTF, human serum transferrin; [Bi(Hcit)], Bi(III) citrate; HSA, human serum albumin; HSQC, heteronuclear single-quantum coherence; hTF/2N, recombinant N-lobe of hTF; NTA, nitrilotriacetate; pH*, pH meter reading in D2O; RBC, ranitidine bismuth citrate (an amorphous solid containing ranitidine, bismuth, and citrate in an approximate 1:1:1 molar ratio); ranitidine, N,N-dimethyl-5-(3-nitromethylene-7-thia-2,4-diazaoctyl)furan-2-methanamine; rHA, recombinant human albumin.
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