(Received for publication, December 12, 1995; and in revised form, February 8, 1996)
From the
Large metal ions (>0.9 Å ionic radius) have previously
been found to bind only weakly to human serum transferrin (hTF, 80
kDa), presumably because the interdomain cleft cannot close around the
metal and synergistic anion. Surprisingly, therefore, we report that
Bi (ionic radius 1.03 Å), a metal ion widely
used in anti-ulcer drugs, binds strongly to both the N- and C-lobes
with log K
* = 19.42 and log K
* = 18.58 (10 mM Hepes, 5 mM bicarbonate, 310 K). The uptake of Bi
by apo-hTF
from bismuth citrate complexes is very slow (hours), whereas that from
bismuth nitrilotriacetate is rapid (minutes). Evidence from absorption
and NMR spectroscopy is presented to show that Bi
binds to the specific Fe
binding sites along
with carbonate as the synergistic anion. Under the conditions used,
preferential binding of Bi
to the C-lobe of hTF is
observed. Linear free energy relationships show that there is a strong
correlation between the strength of binding of Bi
and
Fe
to a wide variety of ligands which include
transferrin. Therefore we conclude that the strength of metal ion
binding to transferrin is determined more by the ligand donor set than
by the size of the ion.
Transferrin (80 kDa) is a serum iron transport glycoprotein with
a concentration in blood of 2-4 mg/ml. Its normal function
is to carry iron as Fe
between sites of uptake,
utilization, and
storage(1, 2, 3, 4) . It contains
two specific Fe
binding sites per molecule, each
approximately octahedral and consisting of two tyrosinates, one
histidine, one aspartate, and a bidentate carbonate anion (the
synergistic anion) derived from the
buffer(5, 6, 7) . Iron is situated in N-lobe
and C-lobe interdomain clefts, which close around the metal and
synergistic anion(4) . Apo-hTF (
)is known to bind to
a wide variety of metal ions. The strength of binding appears to be
dependent on the size of the metal ion, being optimum for
Fe
(ionic radius 0.65 Å), weaker for either
slightly smaller, e.g. Ga
(0.62 Å), or
slightly larger, e.g. In
(0.80 Å),
ions and much weaker for very small, e.g. Al
(0.54 Å), or very large, e.g. lanthanide
(0.86-1.03 Å) ions (Table 1). These data appear to
indicate that strong binding arises from the matching of the ionic
radius to the size of the binding cleft(4) . In the case of the
larger ions, the interdomain cleft may not close at all.
Since
transferrin is only about 30% saturated with iron in normal
serum(8) , there is substantial binding capacity for other
metal ions that enter the blood. Thus transferrin is an important serum
transport agent for metal ions of therapeutic, diagnostic or toxic
importance including Al(9) Ga
,
In
(10) , and
Ru
(11) .
There are no previous reports of
the binding of Bi to transferrin, although such
binding might be expected to be weak on account of the large size of
Bi
(ionic radius 1.03 Å, log K
5; predicted from Table 1). Bismuth compounds have been used in
medicine for more than 200 years for a variety of gastrointestinal
disorders(12, 13) . There is particular interest in
bismuth(III) citrate solubilized by ammonium and potassium hydroxide,
which forms the basis of the colloidal bismuth subcitrate present, for
example, in the drugs Telen
(Byk Gulden) and
De-Nol
(Gist Brocade)(14, 15) .
Recently a new adduct of ranitidine with bismuth citrate (Glaxo
Wellcome plc), which combines the antisecretory action of ranitidine
with mucosal protectant and bactericidal properties of bismuth(III),
has been granted a product license in the UK(16, 17) .
There is also interest in using compounds containing radioactive
bismuth isotopes as targeted radiotherapeutic agents(18) .
Despite this medicinal interest, the speciation of bismuth in blood
plasma, in particular the binding of Bi
to blood
plasma proteins, is poorly understood.
In this paper we report the
first detection of the binding of Bi to serum
transferrin(19) . We determine the binding constants via
electronic absorption spectroscopy and show, by absorption and NMR
spectroscopy, that Bi
is taken up into the specific
iron sites of transferrin and is accompanied by concomitant binding of
the synergistic anion carbonate. We also show that there is
preferential uptake into the C-lobe site. Linear free energy plots
correlating the strength of binding of Bi
and
Fe
to a wide variety of ligands provide an insight
into the reasons why Bi
binds tightly to transferrin.
A 50 mM stock solution of [Bi(Hcit)] was prepared by adding the
minimum amount of ammonia solution to a suspension of
[Bi(Hcit)] until it became clear. The final pH of this
solution was 7, and it was then diluted to 1.0 mM before
use.
[Bi(NTA)] solutions were prepared from a
stock solution of [Bi(NTA)] in water (pH
4.7) by adding
the appropriate amount of NTA. The bismuth concentration was determined
by inductively coupled-plasma atomic emission spectroscopy (Perkin
Elmer).
A solution of [Fe(NTA)] was prepared
from an iron atomic absorption standard solution (1000 ppm in 1%
HNO
, Aldrich) and 2 mol eq of NTA. The pH was gently raised
to 5-6 with microliter amounts of NaOH (1 M). This
solution was then further diluted to give the required
[Fe(NTA)
] concentration in 10 mM Hepes
buffer.
Proton-coupled C NMR spectra were recorded on a JEOL
GSX 500 instrument operating at 125 MHz. Typically 10,000-30,000
scans were collected using a pulsewidth of 6 µs (50°),
relaxation delay 2 s, 16,384 data points. The spectra were processed
using exponential functions (line-broadening of 5-8 Hz). The
chemical shift reference was TSP via added internal dioxan (67.4 ppm).
Figure 1:
Dependence of absorbance (A) on
time for a solution containing apo-hTF and 2 mol eq
[Bi(Hcit)] in 10 mM Hepes buffer solution (5 mM NaHCO, pH 7.4) at 310 K. Two new absorbance bands
centered at 241 and 295 nm appear and are indicative of Bi
binding to the specific binding sites of transferrin. Reaction
times from bottom to top: 0, 5, 10, 15, 30, 60, 120, 240, 420, and 660
min.
Figure 2:
Dependence of A on
time for the reaction of apo-hTF with different mol ratios of
[Bi(Hcit)] at 310 K.
Under similar conditions, the reaction of apo-hTF with [Bi(NTA)] also gave rise to the same absorbance bands at 241 and 295 nm, but at a much faster rate than that observed for [Bi(Hcit)]. Complete reaction of hTF with 1 or 2 mol eq of [Bi(NTA)] occurred within 30 min (data not shown).
Figure 3:
Titration curves for addition of
[Bi(NTA)] to 1.09
10
M apo-hTF in 10 mM Hepes buffer at pH 7.4
containing 5 mM bicarbonate, with x = 1, 8,
and 20.
equals the absorbance at 241 nm divided by the
transferrin concentration; r is the ratio of [Bi] to
[hTF].
The slope of the initial
linear portion of these curves is 21,900 (± 650) M cm
and can be equated
to the molar absorptivity of transferrin with one site saturated with
Bi
(
). Hence if the two sites
are equivalent, a
value of
43,800 M
cm
would be expected
when both sites are filled. These values are compared to those of other
metal-transferrin complexes in Table 1.
The absorbance data obtained at different molar ratios of NTA:Bi were used to calculate the binding constants for bismuth-transferrin complexes, using and below.
The relationships between the equilibrium constants
K and K
and the
stability constants for bismuth transferrin (K
and
K
) and bismuth NTA are given by the following
equations.
If it is assumed that the two binding sites for bismuth on hTF are independent and equivalent, then
where the fractional saturation Y =
[Bi-bound]/[hTF], n = average number of bismuth ions bound per transferrin
molecule, K
is the intrinsic binding constant, and K
= 2 K
and K
= K
/2.
For 20:1 NTA:Bi titration curve, the maximum observed
(
18,000) never exceeded the calculated molar absorptivity
(
= 21,900 M
cm
) (Fig. 3), even at r = 2.5. This suggested that only one Bi
ion binds to hTF under these conditions, and so these data were
used to calculate K
. The slope of the
plot of 1/Y versus [NTA]/[Bi(NTA)] gave
log K
= 1.88 ± 0.02
(correlation coefficient r = 0.992, n =
1.01).
From the 8:1 NTA:Bi titration curve, it was possible to use a
similar plot to calculate log K = 1.87 ± 0.02 (in agreement with that obtained from
the 1:20 titration curve) and log K
= 1.28 ± 0.02 (r = 0.99, n = 1.99).
Using the known value (24) for the
stability constant of [Bi(NTA)] of log K=17.75 and and , pH-independent binding constants of log K
= 19.63 and log K
= 19.03 were
calculated for bismuth hTF. However, there is good evidence that the
two binding sites are not in fact equivalent (see below), and therefore
the second binding constant was recalculated using the mass balance
equation, followed by fitting the apparent absorptivity at each point
on the titration curve (the method of Harris and Pecoraro; see
``Experimental Procedures''). This was simplified by the
initial calculation of K
and gave log K
= 18.80 ± 0.05, a value 0.2 log
units smaller than that obtained from the Hill plot (). The
fitting procedure is very sensitive to the value of K
, and hence the difference in binding constants
between the two sites does not arise from experimental errors. This
confirms that the two Bi
sites are non-equivalent.
The difference in binding constants of log K
- log K
= 0.84 is similar to
that reported for Al
, Ga
, and
Fe
(Table 1).
The bicarbonate-independent binding constants (K*) were calculated from the relationship (25) shown by ,
where represents the fractional saturation of
the apo-hTF binding sites with
bicarbonate.
With 5 mM bicarbonate, pH 7.4, = 0.6, giving log K*
= 19.42
and log K*
= 18.58.
Figure 4:
Dependence of absorbance on pH for a
sample of hTF saturated with
Bi.
Figure 5:
Graphs showing the changes in extinction
coefficient at 241 nm of bismuth transferrin with increasing citrate or
NTA (L) concentrations, at 310 K, pH 7.4. This experiment was carried
out by first adding 3.0 mol eq Bi(NTA) to apo-hTF to form
Bi
-hTF, followed by titration with free NTA or citrate.
Each solution was allowed to equilibrate for 30 min before recording
the absorbance.
Figure 6:
Changes in the visible absorption spectrum
after addition of various amounts of [Fe(NTA)] to
a solution of hTF (10 µM) in 10 mM Hepes buffer
pH 7.4, containing 5 mM bicarbonate and 20 mol eq
[Bi(NTA)] (i.e. both metal sites saturated with
Bi
). From bottom to top: 0, 0.5,
1.0, 1.5, 2.0, 2.5, 3.0, and 4.0 mol eq
[Fe(NTA)
]. Solutions were allowed to equilibrate
for 30 min at 310 K after iron addition before the spectrum was
recorded.
Figure 7:
125 MHz C NMR spectra of hTF
(0.9 mM, 50% H
O, 50% D
O, pH* 7.25) in
the presence of 10 mM H
CO
(A), and with addition of 0.89 mol eq
[Bi(NTA)] (B) and after addition of 2 mol eq
[Bi(NTA)] (C).
Figure 8:
Resolution-enhanced 500-MHz H
NMR spectra of apo-hTF (0.5 mM) in 0.1 M KCl, pH*
7.25 in the high field region (A), the aromatic region (B), and N-acetyl region (C), before (bottom) and after addition of 1 (middle) and 2 (top) mol eq of
[Bi(NTA)].
A
two-dimensional TOCSY H NMR spectrum of apo-hTF in presence
of 10 mM bicarbonate, pH* 7.25 showed a cross-peak at
6.34(peak q)/7.72 ppm, suggesting that this may arise from a
C
H/C
H connectivity of the imidazole ring of a histidine
residue. Furthermore, in the His C
H region (7.5-8.8 ppm, Fig. 8B), peaks s`, t`, and j` appear on addition of the first equivalent of Bi
but remain almost unchanged with the second equivalent, whereas peak y decreases in intensity gradually throughout the
titration.
The most intense peaks in the region of the spectrum from
2.0 to 2.2 ppm are likely to arise from the N-acetyl
groups of the glycan chains in the C-lobe of hTF. On addition of the
first equivalent of Bi
to apo-hTF, a new apparent
singlet peak appears at 2.097 ppm, the peak at 2.080 ppm splits into
two, and the shoulder at
2.038 ppm decreases in intensity (see Fig. 8C). These changes occur progressively from 0 to 1
mol eq of Bi
, and no further changes occur in this
region on addition of the second equivalent of Bi
.
There appear to be no previous reports of the binding of
transferrin to bismuth, and it might be expected that Bi would bind only weakly since it is a large metal ion
(six-coordinate ionic radius 1.03 Å), and ions of this size, such
as the lighter lanthanide ions, have previously been found to bind
10
times more weakly than Fe
(Table 1). The concept has arisen that transferrin cannot
exhibit the same closed structure with large metal ions bound because
the interdomain clefts cannot close(4) . Surprisingly,
therefore, our data show that there is strong binding of Bi
to human serum transferrin.
The changes in the UV spectra of
hTF on binding of Bi are similar to those observed
previously for the binding of other metal ions to the specific
Fe
binding sites. The new bands at 241 and 295 nm are
attributable to binding to tyrosine ligands (
-
* transitions).
From the magnitude of the change in extinction coefficient ( Table 1and Fig. 3), it can deduced that two tyrosines are
involved in binding Bi
in both the N- and C-lobes
(Tyr-95/Tyr-188, and Tyr-426/Tyr-517) as is the case for
Fe
. The displacement of Bi
from
transferrin by Fe
, and lack of binding to
Fe
-hTF provided further evidence for specific binding of
Bi
to the protein. Moreover the
C NMR
data show that carbonate is directly bound to Bi
in
each lobe. Therefore Bi
can now be added to
Cr
, VO
, Mn
,
Co
, Cu
, Ga
, and
In
as metal ions that satisfy the criteria (4, 25) for specific metal binding to transferrin.
The two binding constants that we have determined for Bi binding to transferrin are only slightly lower than those for
Ga
binding (Table 1). Several other previous
studies have demonstrated the non-equivalence of the two transferrin
binding sites, e.g. by NMR(27, 28, 29, 30, 31, 32) and
EPR(21, 34) . The strength of Bi
binding to transferrin is not so surprising when a linear free
energy relationship (LFER) is used to compare the binding constants for
Bi
with those of Fe
and a wide
range of O,N-donor ligands, Fig. 9. There is a good correlation,
which is described by , with correlation coefficient r = 0.979.
Figure 9:
Linear free energy relationships (LFER) for the complexation of Bi and
Fe
with oxygen and nitrogen donors. These data were
taken from (24) . The points for the two binding constants of
transferrin are shown as solid circles. CDTA, trans-1,2-diaminocyclohexane; DTPA,
diethylenetriaminepentaacetic acid; ; H
hbdtta, N,N`-bis(2-hydroxybenzyl)diethylenetriamine-N,N`,N"-triacetic
acid; HIMDA, N-(2-hydroxyethyl)iminodiethanoic acid; IDA, iminodiethanoic acid.
The slope of the LFER is greater than 1.0, and many of the
stability constants for Bi complexes are greater than
those of Fe
. However, the oxygen ligands in the plot
are carboxylates and not phenolates, and the former might favor
Bi
since they are softer donors. The two bismuth-hTF
binding constants are roughly within the LFER region, although the
values are 3 log units lower than predicted, i.e. hTF should
bind Bi
more strongly than Fe
.
However, the slope of the LFER might be lower if more appropriate model
complexes containing phenolates could be incorporated. Harris et
al. have found that hydroxybenzyl-containing ligands provide the
best predictors for In-hTF binding constants(25) . The recent
data of Hancock et al.(35) support this argument.
They have found that the binding constants of Bi
and
Fe
with DTPA change from log K
(35.6) > log K
(28.0) to log K
(27.76) < log K
(30.44) when the two terminal carboxylate groups are replaced by
phenolate groups. The strength of binding of metal ions to hTF
therefore appears to be related more to the type of donor set provided
by the protein than to the size of the metal ion.
A critical maximum
metal ion radius of 0.8-0.9 Å has been suggested for domain
closure to occur(36) . Thus it has been suggested that the
interdomain cleft is open when Ce (r = 1.01 Å) binds to lactoferrin. In the case of
Bi
, it seems clear from the values of
( Table 1and Fig. 3) that two Tyr
ligands are bound to Bi
in each lobe, as is the case
with Fe
. Since the Tyr residues are on domain 2 and
an interdomain polypeptide strand, such binding could occur without
domain closure(37) .
It would therefore be interesting to study bismuth hTF by x-ray scattering to determine the radius of gyration for comparison with other metallotransferrins(38, 39) .
The NMR studies show
that (bi)carbonate binds to both the N- and C-lobes of hTF
concomitantly with Bi. The
C shift of
the bound synergistic anion (165.8 ppm) is close to that observed
previously for other metallotransferrins, e.g. 166.0/166.2 ppm
for Tl
(40) , 165.4 ppm for
Al
, and 166.5 ppm for
Ga
(41) , suggesting a similar mode of
binding, probably as a bidentate carbonate ion. The anion is strongly
bound to bismuth hTF and is not readily removed by dialysis. Studies of
Bi
binding to transferrin can also be readily
monitored by
H NMR since apo-hTF and Bi-hTF are in slow
exchange on the NMR time scale (indicative of strong Bi
binding). Peaks in both the aromatic and the methyl regions of
the spectrum suggest preferential binding of Bi
to
the C-lobe. The relatively sharp peak q at 6.34 ppm is
affected by the first mol eq of Bi
and has been
previously assigned to a C-lobe residue(42) . It has an
associated low pK
(5.87) and an unusual pH
titration shift range (0.75 ppm), and arises from a His C
proton
(based on two-dimensional TOCSY data) with an unusual high field shift.
From examination of the x-ray structure of hTF with Fe
in the C-lobe(7) , possible candidates are His-473 or
His-535. The sharp resonances at 2.0-2.1 ppm are attributable to
the N-acetyls of the NAcGlc and NAcNeu residues in each of the
two biantennary glycan chains attached to Asn-413 and Asn-611 in the
C-lobe of hTF. Since these resonances were perturbed only on addition
of the first mol eq of Bi
, we can conclude that
preferential binding occurs to the C-lobe of hTF. A similar NMR
behavior has been observed for Ga
(with oxalate as
synergistic anion) and In
binding to
hTF(28, 43) , and several other metal ions are known
to bind more strongly to the C-lobe of hTF than to the N-lobe (40) .
No attempt was made to investigate the kinetics of
reactions of hTF with Bi citrate or NTA complexes at
this stage, but it is apparent that there at least two distinct kinetic
steps. The citrate complex reacts much more slowly with hTF than the
NTA complex, and dissociation of the low molecular mass ligand bound to
Bi
appears to be partly rate-determining. Similar
observations have been reported for Fe
binding to
hTF(33, 44) .
We have shown that
Bi binds specifically to the Fe
binding sites in transferrin and that (bi)carbonate is also bound
as the synergistic anion. Although Bi
binds
relatively strongly, it can be displaced by Fe
. The
NMR data show that Bi
binds preferentially to the
C-lobe, as has been found for several other metal ions. The ligands
already bound to Bi
play an important role in
determining the rate of transfer of Bi
from low
molecular mass complexes onto transferrin.
It will be of interest
for future work to investigate whether the domains of the N- and
C-lobes close around the large Bi ions when the metal
is bound, and to determine whether Bi
binding occurs
in intact blood plasma or other fluids and whether transferrin plays
any role in determining the biodistribution of bismuth in the body.