©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Periodate Modification of Human Serum Transferrin Fe(III)-binding Sites
INHIBITION OF CARBONATE INSERTION INTO Fe(III)- AND Cu(II)-CHELATOR-TRANSFERRIN TERNARY COMPLEXES (*)

David C. Ross, Timothy J. Egan, and Langley R. Purves

From the (1) Department of Chemical Pathology, University of Cape Town, Red Cross War Memorial Children's Hospital, Rondebosch 7700, Cape Town, South Africa

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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)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.


INTRODUCTION

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.


MATERIALS AND METHODS

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.

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.

Diferric transferrin was prepared by the addition of 2.2 equivalents of Fe(III)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.

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.

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) .

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

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) .


RESULTS

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)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.

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)NTA will be more efficient at the former site, which may distort the kinetic information.

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)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.

On-line formulae not verified for accuracy

The calculated extinction coefficients for (Fe(NTA)) complexes of periodate-modified transferrin are 1.6 10M cm and 1.5 10M 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) .

The binding constants determined here for periodate-modified transferrin are very similar to those reported for Fe(III)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.

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)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)).

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. 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.

The effects of various anions on the absorption spectrum of the Fe(III)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.

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) .


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.

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.


DISCUSSION

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)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.

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)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.

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/HO 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) .

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)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.

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)NTA ternary complex of periodate-modified transferrin.

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.


FOOTNOTES

*
This work was supported by the University of Cape Town Staff Research Fund and the South African Medical Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; NTA, nitrilotriacetate; LMCT, ligand to metal charge transfer; EPR, electron paramagnetic resonance.


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