From the
Periodate modification of human serum transferrin produces a
species that binds Fe(III) weakly at pH 7.4 contrary to previous
reports that Fe(III)-binding activity is completely lost. Ternary
complexes of periodate-modified transferrin and either Fe(III) with
nitrilotriacetate (NTA), oxalate, citrate, or EDTA, or of Cu(II) with
oxalate could be formed. Peak wavelength maxima of these spectral bands
are identical to those reported for native transferrin in the absence
of bicarbonate. No carbonate ternary complexes of periodate-modified
transferrin with Fe(III), Al(III), Cu(II), or Zn(II) could be formed.
Conditional (Fe(NTA)) binding constants (log K) for C- and
N-terminal modified sites are 7.33 and 7.54, respectively. The
respective extinction coefficients at 470 nm are decreased 45% compared
with the native protein. The electron paramagnetic resonance spectrum
of the complex closely resembles that of the Fe(III)
Transferrin and the two closely related proteins, lactoferrin
and ovotransferrin, bind two Fe(III) ions at sites in the N- and
C-terminal lobes of the polypeptide together with two carbonate ions
known as the synergistic anions
(1, 2, 3) . Each
binding site of transferrin supplies four protein-based Fe(III)
ligands, aspartic acid, histidine, and two tyrosines
(4) , with
the remaining two co-ordination sites occupied by
carbonate
(5, 6, 7) , which interacts with the
protein at a positively charged region involving an
arginine
(5, 7) . Bicarbonate binding to the protein is
suspected to preceed Fe(III) binding, preparing the site for the
metal
(8, 9) . Physical
(10, 11, 12) and crystallographic (13-15) studies indicate a major
conformational change upon Fe(III) binding.
Transferrin can bind a
variety of metal ions (e.g. Fe(III), Al(III), Cu(II)) with
variable affinity in the presence of bicarbonate (16, 17). Carbonate is
the naturally occurring synergistic anion and the one that forms by far
the strongest metal complexes with transferrin
(18) . However, a
select group of anions can also act as synergistic anions in the
absence of bicarbonate provided they conform to certain size
limitations, and that they contain a carboxylate group to interact with
the protein and some Lewis base atom (e.g. oxygen, nitrogen,
or sulfur) to interact with the
metal
(7, 18, 19, 20, 21, 22) .
Transferrin has been implicated in binding and mobilizing aluminium
and possibly other metals causing pathological processes
(23) .
Furthermore, transferrin is used to carry metals like gallium and
indium for tissue-imaging purposes
(24, 25) . Study of
the thermodynamics and kinetics of both metal binding and release from
transferrin is therefore of relevance. Due to uncertainties when
analyzing multicomponent curves, particularly when individual sites
respond differently to perturbing factors, monosited transferrins would
be useful
(26, 27, 28) . Interlobe interactions
might modulate iron release as reported for lactoferrin
(29, 30) and ovotransferrin
(31) , leaving the possibility
that ``half-molecules'' of transferrins might have
limitations in this regard. Therefore it is of interest to devise
methods of preparing mono-metallated transferrin species.
In this
study we have examined in detail the metal- and anion-binding
properties of human serum transferrin metal-binding sites following
periodate modification. In addition, we have studied the effects of
modification of a single site on the metal-binding properties of and on
the kinetics of Fe(III) release from the unmodified site.
HEPES, EDTA, nitrilotriacetic acid, tetrasodium
pyrophosphate, and sodium metaperiodate were obtained from Sigma.
Ferric chloride and acrylogel were purchased from BDH Chemicals, Ltd.
All other chemicals were of analytical reagent grade.
Diferric transferrin was prepared by the addition of
2.2 equivalents of Fe(III)
Removal of Fe(III) from transferrin was performed
by dialysis of the protein solution against 50 mM citrate, pH
4.5, followed by dialysis against water. To remove contaminating ions
(e.g. NTA, citrate, or periodate) from transferrin, the
protein solution was dialyzed against 0.1 M sodium perchlorate
followed by 0.1 M sodium chloride and finally against water.
These dialysis solutions were maintained at neutral pH by addition of
small aliquots of ammonium hydroxide solution. Protein was either
lyophilized or else kept in 5 mM HEPES pH 7.4 at 4 °C.
The value of log K for
(Fe(NTA)) binding to each modified site of periodate-modified
monoferric transferrins was then calculated for each point using
Equation 1.
On-line formulae not verified for accuracy
The reaction of sodium metaperiodate with apotransferrin is
shown in Fig. 1. Visible absorption spectra of transferrin in the
presence of 2.8 equivalents of Fe(III) provided as Fe(III)
Very little of the N-terminal monoferric- species
forms during the reaction indicating heterogeneity in site reactivity,
with the vacant N-terminal site being more susceptible to modification.
This result was confirmed by monitoring the periodate reaction with the
monoferric transferrins (results not shown) and is consistent with the
biphasic kinetics observed in Fig. 1A. As formation of
the Fe(III)-carbonate ternary complex of native transferrin at the
C-terminal site is faster than at the N-terminal site
(51) ,
quenching of the periodate reaction by Fe(III)
Solution spectra of periodate-modified transferrin in
the presence of various Fe(III)-chelate complexes are shown in
Fig. 2
. The dashedline is the spectrum of a
periodate-modified transferrin solution at ambient carbon dioxide
saturation (0.14 mM bicarbonate) in the presence of a 2-fold
molar excess of Fe(III) chloride. The solution is yellow, similar to
that described for the mixture of Fe(III) with transferrin in the
absence of bicarbonate
(18, 52) . No LMCT band was
evident, even when the bicarbonate concentration was raised to 20
mM. Inclusion of a 1.25 molar excess of NTA, oxalate, citrate,
or EDTA over the Fe(III) prior to the addition to the transferrin
solution results in orange (oxalate), pink (NTA and citrate), or purple
(EDTA) colored solutions. NTA produced by far the most intense
absorption band under these conditions. The spectra show distinct LMCT
bands with peak maxima ranging from 470 nm for NTA and 485 nm for
oxalate to 523 nm for EDTA. These values are almost identical to those
reported for native transferrin with these synergistic anions in the
absence of bicarbonate
(7, 18, 19, 22) .
Citrate, which has been reported to be
nonsynergistic
(19, 22, 53, 54, 55) ,
gave a wavelength maximum of 493 nm. No LMCT bands were evident with
the synergistic anions maleate, glycine, or thioglycollate under these
conditions. These results suggest that of the potential synergistic
anions for Fe(III) binding to native transferrin, only those that are
strong Fe(III) chelators can effectively form ternary complexes in the
periodate-modified protein. The Fe(III)
On-line formulae not verified for accuracy
The calculated extinction coefficients
for (Fe(NTA)) complexes of periodate-modified transferrin are 1.6
The binding constants determined here for
periodate-modified transferrin are very similar to those reported for
Fe(III)
Further evidence that carbonate does not act as a synergistic anion
in the Fe(III) complex of periodate-modified transferrin is provided by
EPR spectroscopy. The EPR spectrum of native diferric transferrin with
carbonate as synergistic anion (spectrum A) and of a sample of
periodate-modified transferrin with two equivalents of Fe(III)
When an aliquot
of an acidic Fe(III)-citrate stock solution (21.8 mM Fe(III)
chloride in 10 mM HCl and 27.3 mM trisodium citrate,
pH 2) was added to periodate-modified apotransferrin to yield a final
0.4 mM Fe(III) and 0.5 mM citrate solution at pH 7.4,
an LMCT band at 493 nm appeared and increased in intensity up to 2 min.
Thereafter, the intensity of the band diminished to a new, much lower
equilibrium position, and the rate constant for the monoexponential
disappearance of the band was measured as 0.022 min
The effects of various
anions on the absorption spectrum of the Fe(III)
Anion binding by the apo form of the
modified protein was demonstrated by ultraviolet difference
spectroscopy, with a negative difference peak around 245 nm probably
due to stabilization of the protonated forms of the binding site
tyrosyl groups
(46) . Fig. 6shows typical difference
spectra of apotransferrin and periodate-modified apotransferrins in the
presence of pyrophosphate, which is known to bind with high affinity to
apotransferrin, log K around 6 (66), and at 3 mM
should saturate the sites. Periodate-modification resulted in
approximately a 3-fold decrease in the negative peak at 245 nm, while
the peak for transferrin modified only at the N-terminal site lies
halfway between the two. The apotransferrin periodate modified at the
C-terminal site gave a similar spectrum (not shown). Similar spectra
were obtained with bicarbonate, sulfate, and nitrilotriacetate,
although each exhibited slightly different spectra. The intensity of
the negative peak at 295 nm appeared unaffected by
periodate-modification, suggesting this peak may arise from a
tryptophanyl residue
(67) .
The unmodified sites still bind metals (e.g. Fe(III),
Al(III), Cu(II), and Zn(II)) with carbonate as synergistic anion, and
this allows for preparation of mono-metallated transferrins. Rate
constants for Fe(III) release from these unmodified sites to
pyrophosphate were found to be almost identical to those determined for
the native monoferric transferrins.
Periodate-modified transferrin iron binding-sites are shown
to bind Fe(III) or Cu(II) only in the presence of an appropriate
chelating anion, e.g. oxalate or NTA, apparently with
carbonate unable to participate as a synergistic anion. EPR
spectroscopy confirms that the Fe(III)-carbonate ternary complex does
not form when Fe(III)
Previous reports on periodate-modification of transferrin
and ovotransferrin state that iron-binding activity is
lost
(36, 38, 68, 69, 70) . That
urea-PAGE seemingly confirms this can be ascribed to the failure of the
Fe(III)
Periodate oxidation of transferrin
results in linkage of each of the two favorably aligned tyrosyl
residues in each Fe(III)-binding site to form a dityrosine
product
(71) , similar to those produced by ozone or horseradish
peroxidase/H
The
identical wavelength maxima of the various ternary complexes of Fe(III)
and Cu(II) with periodate-modified and native transferrin suggest that
both phenolates ligate the Fe(III), although to be certain of this it
would be necessary to compare the spectra with a comparable biphenolate
complex. Apparently conjugation does not extend between the two
aromatic rings following periodate-induced cross-linking, since this
would cause a shift in the position of the LMCT band
(74) .
Steric factors thus ensure that the aromatic rings of the biphenolate
are not coplanar. The estimated equilibrium constants for (Fe(NTA))
binding to the periodate-modified sites are very similar to the values
reported for Fe(III)
Joining the two phenolate ligands by the cross-link
would result in formation of a seven- or nine-membered metal chelate
ring. This introduces constraint into the complex, probably leading to
poorer orbital overlap between the ligands and the metal and hence to a
decrease in molar absorptivity of the LMCT band
(74) . This most
probably accounts for the observed 45% decrease in extinction
coefficient of the Fe(III)
Perhaps the most striking effect of
periodate oxidation on the site is loss of activity of the
physiological anion, carbonate, for Fe(III) binding. In native
transferrin, it forms by far the strongest complex, due to the ability
of the protein to form a network of hydrogen bonds to the anion and
across the domains of each lobe
(7) . Apparently with other
bulkier synergistic anions this arrangement of stabilizing hydrogen
bonds is weakened due to the poorer fit. Fe(III) binding to native
transferrin is believed to proceed via binding of the Fe(III)-chelate
complex to transferrin followed by carbonate insertion into the Fe(III)
inner coordination sphere, accompanied by loss of the chelator, and
finally closure of the protein over the metal binding site to form a
strong
complex
(18, 31, 51, 75, 76) . It
would thus appear that this crucial carbonate insertion and protein
conformational change is prevented by the cross-link.
Anions such as
chloride, sulfate, and bicarbonate are able to displace the weakly
bound Fe(III) from the ternary complex of periodate-modified
transferrin with NTA. Analogously, bicarbonate can displace weakly
bound Fe(III)-EDTA from native transferrin, although in that case
subsequent formation of the Fe(III)-carbonate-transferrin derivative
occurs
(20) , and phosphate can readily remove the Fe(III) from
the weak Fe(III)-glycine ternary complex
(21) . A variety of
anions are also able to destabilize the weak ternary carbonate
complexes of native transferrin with divalent metals like
Zn(II)
(44) , Cu(II)
(77) , and Cd(II)
(78) . In
these cases, anion displaces the weakly bound metal complex from
transferrin by competing for the metal-binding site. The displacement
of the metal-chelator complexes from periodate-modified transferrin can
in part be accounted for by the observation of anion (e.g. pyrophosphate, sulfate, and bicarbonate) binding at the modified
sites. Since the ability of anions to displace these complexes is not
directly correlated to their strength of binding to the modified
metal-binding sites, it would appear that interaction with other
anion-binding regions (e.g. the site proposed in Ref. 47) on
the protein may also play a role. The inability of bicarbonate to act
as a synergistic anion for metal binding by periodate-modified
transferrin cannot be ascribed to inhibition of bicarbonate binding
prior to formation of the Fe(III)-carbonate-transferrin complex.
The
efficacy of the periodate reaction with the binding-site tyrosyl groups
suggests a specific interaction of this anion with the binding sites.
Due to ultraviolet absorption by periodate and iodate, we were unable
to determine their extent of binding. The binding strength exhibited by
native apotransferrin to oxoanions follows the series pyrophosphate
> phosphate > arsenate > sulfate > bicarbonate > nitrate
and perchlorate
(46, 66, 79) , which is similar
to the series for inhibition of the periodate reaction
(70) . The
central atom of the oxoanion in this sequence follows the series of
increasing electronegativity, phosphorus < arsenic, iodine <
sulfur < carbon < chlorine < nitrogen, all of which are less
electronegative than oxygen. The charge density on the oxygen atoms is
greater when the central atom is of lower electronegativity, and this
would favor their interaction with nucleophiles, Lewis acids, or
hydrogen-bond donors (in this case the protons of the phenolate
groups). We can predict that periodate binding strength will lie
somewhere between that of arsenate and sulfate, i.e. log K around 4, and could be referred to as an affinity reagent.
Periodate-modification of transferrin is shown to have little
significant effect on the rates of Fe(III) release from either of the
unmodified sites to pyrophosphate. Thus this method can be employed to
generate single-sited transferrin molecules to study binding and
release of metal ions like Al(III), Cu(II), Ga(III), Pu(IV), etc. from
the ternary carbonate complexes.
Although periodate modification of
iron-free binding sites of transferrin is not of physiological
relevance, it appears that the dityrosine product is similar to that
formed by the oxidative effects of reactive oxygen species on other
proteins
(72, 73, 80) . Similar protein species
could be produced by oxidative damage to transferrin. If this is the
case, periodate modification models such damage.
NTA ternary
complex formed with native transferrin in the absence of bicarbonate.
Anions, including bicarbonate, at high concentrations destabilize
formation of this Fe(III)
NTA ternary complex, while Fe(III)
chelators readily remove the bound Fe(III). Bicarbonate, sulfate, and
pyrophosphate still bind to the modified binding sites in the absence
of metal although with slightly lower affinity and with lower molar
difference absorptivities. Results are interpreted as an inhibition of
a crucial protein conformational change by an intramolecular
cross-link, preventing formation of the particularly stable
metal-carbonate ternary complex from the less stable metal-chelate
ternary complex. The method can be used to produce monosited
transferrins.
Preparation of Transferrin
Human serum transferrin was
purified from outdated blood bank samples of whole blood by standard
methods, except that the transferrin was purified on a Boehringer
Mannheim zinc chelate affinity adsorbent column
(32) . The apo
form of transferrin was prepared by dialysis of the protein solution to
remove Fe(III) and other ions as outlined below. The protein was
characterized by SDS-polyacrylamide gel electrophoresis
(PAGE)(
)(33) and was usually greater than
98% in purity.
NTA (1:4) at pH 7.4 with ambient carbon
dioxide saturation. After 1 h of equilibration, the protein solution
was dialyzed to remove contaminating ligand ions as outlined below. C-
and N-terminal monoferric transferrins were prepared as described
elsewhere
(26) . They were characterized by urea/EDTA PAGE
(urea-PAGE)
(34) (1 mM EDTA was included in the gel and
in the sample mix) and were usually greater than 90% in purity.
Extinction coefficients used to determine transferrin solution
concentrations were 4860, 2780, and 2080 M
cm
for diferric-, C-terminal monoferric-, and
N-terminal monoferric transferrins respectively at 465 nm or 87,000 and
114,000 M
cm
for apo-
and diferric transferrin, respectively, at 280 nm
(35) . All
spectroscopic measurements were performed either on a Varian DMS 100
spectrophotometer or on a Beckman DU 7500 diode array
spectrophotometer.
Periodate Treatment of Transferrin
Periodate
treatment of apotransferrin (typically 1-10 mg/ml) was performed
for 1 h at 23 °C in the dark in 50 mM HEPES buffer pH 7.4
with 5 mM (unless otherwise stated in the legends to the
figures) freshly prepared sodium metaperiodate. Aliquots for analysis
by urea-PAGE were first mixed with a 5-fold excess of Fe(III)NTA
(1:4) prior to loading onto the gel. Visible absorption spectra of
transferrin solutions (in 50 mM HEPES pH 7.4) were recorded
between 650 and 350 nm following addition of an excess of
Fe(III)
NTA (1:4), as outlined below. In some cases, modified
transferrin solutions were dialyzed to remove contaminating ions as
outlined above. Transferrin modified at the C- or N-terminal sites
only, or at both sites, were prepared by reacting N- or C-terminal
monoferric transferrins or apotransferrin, respectively, with periodate
as described above. As control specimens, diferric transferrin was
treated with periodate as described above and was found to be
effectively protected against modification, as previously
reported
(36, 37, 38) .
Fe(III) Binding to Transferrin
Binding constants
for Fe(III)NTA with modified C- and N-terminal sites were
obtained by titrating periodate-modified N- and C-terminal monoferric
transferrins with Fe(III)
NTA (1:4). Sample (typically 0.2
mM transferrin species) and reference cuvettes were titrated
with Fe(III)
NTA (1:4). Titrations were performed in a
thermostatted cell holder at 20 °C and in 0.1 M sodium
nitrate and 43 mM HEPES pH 7.4 with and without inclusion of 5
mM sodium bicarbonate. The proportion of Fe(III) bound to the
modified transferrin was determined from the absorbance at 470 nm,
using (A -
A
)/(A
-
A
), for each titration point. Knowing the total
Fe(III) concentration, the concentration of Fe(III) not bound to the
site could thus be determined. Making the assumption that the remaining
Fe(III) does not interact with the protein and that one equivalent of
NTA is bound per equivalent of Fe(III), the species distribution for
unbound Fe(III)
NTA was calculated with the program
ESTA
(39) . The pK
values of the
species NTAH
, NTAH
,
NTAH
, and NTAH
were
obtained from Martell and Smith
(40) , as were the formation
constants for the species (FeNTA),
(Fe(NTA)
)
,
(Fe(NTA)(OH))
, (Fe(NTA)(OH)
)
and (Fe(NTA)(OH))
. Formation
constants for (Fe(OH))
,
(Fe(OH)
)
, and (Fe(OH)
) were
from Baes and Mesmer
(41) .
Speciation of Citrate at pH 2.0 and 7.4
The
relative proportions of the various Fe(III)-citrate species present at
pH 2.0 and 7.4 were calculated using the program ESTA
(39) . The
pK values of the species
CitH
, CitH
, and
CitH
were from Martell and Smith
(42) . The
formation constants for the species (FeCit),
(Fe(Cit)
)
, (Fe(CitH))
,
and (Fe(Cit)(OH))
were obtained from
Martin
(43) . The formation constants for the three
Fe(III)-hydroxo species as mentioned above were from Baes and
Mesmer
(41) .
Al(III) and Zn(II) Binding by Transferrin
Al(III)
and Zn(II) binding by transferrin was determined by difference
ultraviolet spectroscopy essentially as previously
reported
(44, 45) . Native apotransferrin or the apo
forms of transferrin modified either at a single site, or at both
sites, were incubated at 0.014 mM in 50 mM HEPES
buffer pH 7.4, 25 °C, with either ambient (0.14 mM) or 5
mM bicarbonate in both sample and reference cuvettes. The
sample cuvette was titrated with Al(III) chloride or Zn(II) acetate
stock solutions, and scans were performed from 230 to 330 nm.
Anion Binding by Transferrin
Anion binding by
transferrin was determined by ultraviolet difference spectroscopy
essentially as previously reported
(46) . Native apotransferrin
or the apo forms of transferrin modified either at a single site or at
both sites, were incubated at 0.014 mM in 50 mM HEPES
buffer pH 7.4, 25 °C, at ambient (0.14 mM) bicarbonate
concentration in both sample and reference cuvettes. The sample cuvette
was titrated with sodium pyrophosphate, sodium sulfate, or sodium
bicarbonate stock solutions, and scans were performed from 240 to 320
nm.
Electron Paramagnetic Resonance
Spectroscopy
Electron paramagnetic resonance (EPR) spectra were
obtained in precision quartz EPR sample tubes (Wilmad) on a Varian E-9
EPR spectrometer. Experimental conditions are given in the legend to
the figure. We acknowledge Dr. P. Aisen, Department of Physiology and
Biophysics, Albert Einstein College of Medicine, Bronx, NY, for making
available his EPR spectrometer. Acquisition of these spectra was
supported in part by Grant DK 37927 from the National Institutes of
Health, United States Public Health Service.
Iron Release from Ferric Transferrins to
Pyrophosphate
Iron release from ferric transferrins to
pyrophosphate was carried out as described previously
(47) .
NTA
(1:4), after treatment of apotransferrin with 5 mM periodate
at pH 7.4 and at ambient carbon dioxide solution saturation (about 0.14
mM bicarbonate), are shown for various reaction times in
Fig. 1A. The visible band centered around 465 nm does
not disappear but merely weakens and appears to red-shift to around
470-475 nm. Since this absorption band is characteristic of
specific binding of Fe(III) to transferrins, resulting from a
(phenolate to Fe(III)) ligand to metal charge-transfer (LMCT)
transition
(48, 49) , it would seem that Fe(III) still
binds to the modified tyrosines. The reaction appears distinctly
biphasic, with one site reacting more rapidly than the other
(Fig. 1A, inset), and the final product
exhibits about half the band intensity compared with the initial
transferrin species.
Figure 1:
Reaction of periodate with
apotransferrin. Apotransferrin (0.049 mM) was incubated in 50
mM HEPES pH 7.4 at 23 °C in the dark with either 2
mM (A) or 5 mM (B) sodium
metaperiodate. At timed intervals, an aliquot of the reaction mix was
removed and the reaction terminated by addition of 0.137 mM
(A) or 0.245 mM (B) Fe(III)NTA (1:4).
Samples were either scanned from 350 to 600 nm (A) or else
subjected to urea-PAGE analysis (B). In (A), the time
intervals are 0 (a), 0.5 min (b), 2 min (c),
7 min (d), 22 min (e), and 240 min (f), and
the inset is a plot of absorbance at 470 nm versus time. In (B), laneM is a marker lane
containing the four species of transferrin, a refers to
apotransferrin, c to C-terminal monoferric transferrin, n to N-terminal monoferric transferrin, and d to diferric
transferrin; laneA is
apotransferrin.
Analysis of the reaction by urea-PAGE is shown
in Fig. 1B. This electrophoretic method relies on the
property of transferrin that binding of Fe(III) (with carbonate) to a
site on the protein renders that lobe of the protein resistant to
denaturation by urea and hence distinguishable from an Fe(III)-free
site
(34, 50) . Thus, the different Fe(III)-transferrin
species have different mobilities in the gel system. 1 mM EDTA
is included in the gel solutions to chelate extraneous iron. Since
excess Fe(III)NTA is added to stop the periodate modification
reaction by loading unmodified sites, the unmodified transferrin will
appear as the diferric species (see time 0 in Fig. 1B).
A small amount of C-terminal monoferric transferrin is present at time
0 and is probably due to some loss of iron from the more labile
N-terminal site during sample preparation and electrophoresis,
particularly with excess NTA present. As the reaction proceeds,
intermediate monoferric species arise due to modification of
transferrin at one site only, until finally as the reaction approaches
completion, the transferrin appears as the apo form. Thus, the modified
sites are incapable of retaining Fe(III) under the conditions of
electrophoresis (1 mM EDTA and 6 M urea), while the
unmodified sites appear unaltered. Periodate-modified transferrin has
the same mobility in the gel as native apotransferrin, indicating that
the modification reaction does not significantly change the net charge
of the protein.
NTA will be more
efficient at the former site, which may distort the kinetic
information.
EDTA ternary complex of
periodate-modified transferrin could even be lyophilized to a purple
solid. Nevertheless, these complexes are weak. Significant color loss
occurred following passage of the Fe(III)
NTA complex of
periodate-modified transferrin through a Pharmacia PD-10 desalting
column.
Figure 2:
Absorption spectra of periodate-modified
apotransferrin in the presence of different Fe(III) sources.
Periodate-modified apotransferrin (0.2 mM) in 50 mM
HEPES pH 7.4, was mixed with (- - -) Fe(III) chloride
(0.4 mM) alone, or else including 0.5 mM NTA
(a), oxalate (b), citrate (c), or EDTA
(d) with the Fe(III). The solutions were then scanned from 350
to 650 nm.
Titrations of the periodate-modified vacant N- and
C-terminal sites of C- and N-terminal monoferric transferrins with
Fe(III)NTA, monitored at the 470-nm wavelength maximum, in the
presence of 0.14 or 5 mM bicarbonate were typical of weak
binding. The data was consistent with binding of one NTA together with
each Fe(III) (Equation 2). The average values of log K obtained for the reaction are 7.33 (S.D. ± 0.08, n = 31) and 7.54 (S.D. ± 0.1, n = 31)
for the modified C- and N-terminal sites respectively.
10
M
cm
and 1.5
10
M
cm
for the periodate-modified N- and
C-terminal sites, respectively. The sum of these extinction
coefficients are about 64% and 55% of those reported for the carbonate
and NTA ternary complexes of native diferric transferrin
respectively
(19) .
NTA binding to native transferrin and ovotransferrin in
the absence of bicarbonate
(31, 51) and differ markedly
from strong binding exhibited by the native proteins in the presence of
bicarbonate (log K values around 20
(57) ). Inclusion of
bicarbonate to 5 mM did not significantly alter the values of
the conditional binding constants obtained in the present study. These
results suggest that the carbonate derivative cannot form, and Fe(III)
binding requires the presence of a chelating ligand, e.g. NTA.
NTA
(1:4) at ambient carbon dioxide saturation (spectrum B) are shown in
Fig. 3
. From the visible absorbance at 470 nm, the latter sample
was estimated to be 69% Fe(III) saturated. Spectrum B is similar to the
EPR spectrum of the Fe(III)
NTA ternary complex of native
transferrin, and does not possess the distinctive peaks associated with
the ternary complex of native transferrin with Fe(III) and
carbonate
(18) .
Figure 3:
EPR spectra of (A) native
diferric transferrin and (B) periodate-modified transferrin in
the presence of two equivalents of Fe(III)NTA (1:4). Protein
concentration was 0.104 mM in both cases, 50 mM HEPES
pH 7.4. EPR conditions: temperature, 77 K; modulation frequency, 100
kHz; modulation amplitude, 10 gauss; time constant, 1.00 s; microwave
frequency, 9.10 GHz; power, 10 mW; gain, 1
10
.
The pH profile for the complexation of
Fe(III)NTA by periodate-modified transferrin as visualized by the
LMCT band at 470 nm was bell-shaped, with a maximum occurring at pH
7.4, while no complex forms below about pH 5 and above pH 9.5 (results
not shown), the solutions being yellow and not pink. Loss of binding at
acidic pH parallels the acid sensitivity of the carbonate and oxalate
ternary complexes of Fe(III) with native
transferrin
(58, 59, 60) . The Fe(III)
NTA
complex of periodate-modified transferrin is, however, far more
sensitive to alkaline pH than the carbonate ternary complex of Fe(III)
with native transferrin
(18, 61) . The speciation model
indicates that this is due to formation of the Fe(III)
NTA hydroxo
species, (Fe(NTA)(OH))
, (Fe(NTA)
(OH)
)
, and
((Fe(NTA)(OH))
, at alkaline pH, lowering
the concentration of the binding species (Fe(NTA)).
.
This is in accordance with the suggestion of a possible
Fe(III)-citrate-native transferrin ternary complex
(62) .
Calculation shows that the predominant Fe(III) species in the initial
acidic Fe(III)-citrate stock solution is (Fe(Cit)) at 79%, with some
(Fe(Cit)(OH))
present (13%). After dilution into the
protein solution at pH 7.4, the model indicates that the predominant
species is (Fe(Cit)(OH))
at 97%, although it is
probable that µ-oxo polymeric Fe(III)-citrate species also form
(not accounted for in the model). It is likely that the (Fe(Cit))
species initially present binds to periodate-modified binding-sites
producing an LMCT band at 493 nm, and thereafter hydrolysis of this
species to (Fe(Cit)(OH))
or µ-oxo polymers
results in dissociation of the Fe(III) from the site. This could
explain why citrate has in the past been considered ineffective as a
synergistic anion for native transferrin.
NTA complex of
periodate-modified apotransferrin are shown in Fig. 4. The anions
markedly decreased the intensity of the LMCT band. Bicarbonate was
found to have an identical effect (not shown). Phosphate appeared the
most effective in this regard, followed by bicarbonate, nitrate,
acetate, sulfate, perchlorate, and chloride. EDTA or pyrophosphate
(Fe(III) chelators) added to 5 or 1 mM, respectively, were
sufficient for rapid and complete color loss of the mixture (not shown,
but spectra similar to that produced by phosphate). Thus, anions
(e.g. nitrate, sulfate, chloride, etc.) destabilize the weak
Fe(III)
NTA ternary complex of periodate-modified transferrin, and
bicarbonate acts as an inhibitor of and not as a synergistic anion for
Fe(III) binding. Anionic chelators (e.g. phosphate,
pyrophosphate, and EDTA) can readily remove the protein-bound Fe(III).
Figure 4:
Effect of anions on the absorption spectra
of periodate-modified transferrin in the presence of Fe(III)NTA.
To periodate-modified apotransferrin (0.1 mM) in 50
mM HEPES pH 7.4 was added 0.25 mM Fe(III)
NTA
(1:4) and 0.2 M anions of sodium salts where indicated, and
then the sample was scanned from 350 to 650 nm. a, no added
anion; b, chloride; c, sulfate; d,
perchlorate; e, acetate; f, nitrate; and g,
phosphate.
The absorption spectrum of periodate-modified apotransferrin in the
presence of a 6-fold molar excess of Cu(II) sulfate, with the inclusion
of a 1.25 molar excess of oxalate over the Cu(II) is shown in
Fig. 5
. The peak centered around 413 nm is similar to the
charge-transfer band of the Cu(II)-oxalate ternary complexes of native
transferrin and lactoferrin
(7, 63, 64) . The
Cu(II)-carbonate complex of native transferrin or lactoferrin exhibits
a peak at around 430-440 nm
(7, 64, 65) .
No similar charge-transfer absorption band was observed when either NTA
or EDTA were substituted for oxalate in the Cu(II) mixture with
periodate-modified apotransferrin, or when bicarbonate was added to 20
mM in the absence of any of these chelating anions. The broad
absorption band around 685 nm is due to
``d-d'' transitions in the Cu(II)-aquo
complex and was present in all solutions containing Cu(II) sulfate.
Figure 5:
Absorption spectrum of periodate-modified
apotransferrin in the presence of Cu(II)-oxalate. Periodate-modified
apotransferrin (0.2 mM) in 50 mM HEPES pH 7.4 was
mixed with Cu(II) sulfate (1.2 mM) and oxalate (1.5
mM), and then the sample was scanned from 350 to 800 nm.
a, Cu(II)-oxalate LMCT band of periodate-modified transferrin;
b, aquo-Cu(II) d-d transition.
Periodate-modified apotransferrin was unable to bind Al(III) or
Zn(II) in the presence of bicarbonate as evidenced by the lack of a
difference absorption peak around 240 nm as measured by ultraviolet
spectroscopy, while the unmodified sites formed stable carbonate
derivatives with both metals.
Figure 6:
Anion binding to the apo form of
peridate-modified transferrins. Transferrin (0.014 mM) either
as periodate-modified apotransferrin (b), apo form of
N-terminal site periodate-modified transferrin (c), or native
apotransferrin in 50 mM HEPES pH 7.4 (d) was mixed
with 3 mM sodium pyrophosphate and then an ultraviolet
difference spectrum of the mixtures obtained from 240 to 330
nm.
Titrations of apotransferrin and
periodate-modified apotransferrin were performed with bicarbonate,
pyrophosphate, and sulfate. The curves show typical saturation binding
characteristics, and were analyzed either as two separate sites or
averaged for a single type of site, as the two methods were largely
indistinguishable. Binding constants (log K) and molar
difference absorptivities for native apotransferrin were 2.6 (3824
M cm
) and 2.2 (3033
M
cm
) for bicarbonate,
3.7 (4517 M
cm
) and 3.2
(4446 M
cm
) for sulfate,
and 5.8 (averaged for both sites) (9800 M
cm
, total) for pyrophosphate. These values are
consistent with previous reports
(46, 66) .
Periodate-modified transferrin bound bicarbonate, sulfate, and
pyrophosphate with average constants of 2.2, 2.7, and 3.5,
respectively, and total molar difference absorptivities of 2486
M
cm
, 2750
M
cm
, and 3200
M
cm
, respectively.
Molar difference absorptivities have decreased to about a third
following modification, with a decrease in the binding constants.
NTA is added to periodate-modified
transferrin. No carbonate derivatives of periodate-modified
apotransferrin and other metals such as Al(III) or Zn(II) could be
formed.
NTA complex to prevent urea denaturation of
periodate-modified transferrin. However, when determining
Fe(III)-binding capacity from chromogenic activity, several factors
affect color generation. These include high pH (>8), which
destabilizes the Fe(III)-chelate complexes of the modified protein by
forming Fe(III)-chelate-hydroxo species, the use of citrate or other
Fe(III) chelators as opposed to NTA, inclusion of high concentrations
of bicarbonate or other anions in the mixture, as well as the use of
phosphate as a buffering agent.
O
in other proteins (72, 73). NMR
studies
(68) indicate loss of protons from tyrosyl aromatic
carbon atoms 2 and 6 (ortho) or possibly from those at positions 3 and
5 (meta) suggesting a 2,2`-, a 3,3`-, or perhaps a hybrid linkage.
Examination of the crystal structure of apolactoferrin (14) indicates
that the distances between the phenoxyl oxygens of the pair of
metal-binding tyrosyl residues of both sites are such that they are
able to hydrogen-bond to periodate. The C-2 atoms of the tyrosyl
aromatic rings are relatively far apart (about 5 Å) and therefore
must be drawn closer together when the dityrosyl product is formed.
This dityrosyl moeity represents an intramolecular cross-link at the
hinge-bending region of each lobe, with one of the pair of linked
tyrosyl residues in contact with the hinge residues
(15) .
NTA binding to native transferrin in the
absence of bicarbonate
(51) . This is consistent with the
spectroscopic evidence, since a change in binding strength is usually
accompanied by a shift in the spectrum
(65) . The most simple
rationalization of this thermodynamic and spectroscopic evidence is
that the role of chelators in the ternary complexes of
periodate-modified transferrin is the same as in the bicarbonate-free
native protein.
NTA ternary complex of
periodate-modified transferrin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.
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Molecular and Cellular Proteomics
Journal of Lipid Research
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