Nonequivalence of the metal binding sites in vanadyl-labeled human serum transferrin.

Vanadyl ion, VO(IV), has been used as an electron paramagnetic resonance (EPR) spin label to study the metal-binding properties of human serum transferrin in the presence of bicarbonate. Iron-saturated transferrin does not bind the vanadyl ion. Room temperature titrations of apotransferrin with VO(IV) as monitored by EPR indicate the extent of binding to be pH dependent, with a full 0.2 VO(IV) ions per transferrin molecule bound at pH 7.5 and 9, but only about 1.2 VO(IV) ions bound at pH 6. The EPR spectra of frozen solutions with or without 0.1 M NaCUO4 at 77 K show that there are two spectroscopically nonequivalent binding sites (A and B) with a slight difference in binding constants. One site (A site) exhibits essentially constant binding capacity in the pH range 6-9, but the other (B site) becomes less avialable as the pH is reduced below 7. Results with mixed Fe(III)-VO(IV) transferrin complexes suggest that iron shows a slight tendency to bind at the B site over the A site pH 7.5 and 9.0. Only the B site in both vanadyl and iron transferrins is perturbed by the presence of perchlorate.     

Vanadium complexes of transferrin and ferritin in the rat

Vanadium associates with serum transferrin of rats administered vanadyl(IV) sulfate or ammonium metavanadate(V) by gastric intubation. Low molecular weight species account for only 3% of the vanadium present in plasma. The element distributes between the two major isotransferrins in proportion to their concentrations. Rat apotransferrin binds both vanadium(IV) and vanadium(V), forming 2:1 metal-protein complexes in both instances. Although the two isotransferrins apparently differ in their physiological properties, they exhibit identical vanadyl(IV) (VO2+) EPR spectra, indicating identical or very similar metal binding sites for both proteins. In contrast to other transferrins, the two sites of the rat protein are spectroscopically indistinguishable and exhibit a VO2+ EPR spectrum similar to that of the C-terminal metal binding site of human serum transferrin. VO2+ EPR signals are observed with liver, spleen, and kidney tissue samples from animals maintained on a vanadium-supplemented diet. These signals arise from a specific intracellular VO2+ complex with the iron storage protein ferritin.     

Metal site conformational states of vanadyl(IV) human serotransferrin complexes.

This study was undertaken to investigate the conformational states of the two metal sites in the human serum transferrin molecule. The 9.2 GHz electron paramagnetic resonance spectra of frozen solutions of divanadyl(IV) transferrin consist of a superposition of two sets of resonances, A and B, due to the magnetically nonequivalent binding environments of the VO2+ ion. Examination of the intensities of the A and B resonances as a function of pH from 6.0 to 10.7 reveals that they arise from two conformational states of the metal sites in which the geometrical arrangement and/or identity of one or more ligands in the first coordination sphere are different. From pH 7.5 to 9.0, the metal sites exist in A and B conformations but above pH 9.0 the A conformation. This transformation is coupled to the ionization of an apparently noncoordinating protein functional group with a pK - 10.0 +/- 0.1. Below pH 7.0, binding in the B conformation is rapidly lost, driven in part by the protonation of a functional group, possibly the anion, with a pK - 6.6 +/- 0.1. In 90% D2O, this pK is elevated to 7.8 +/- 0.1. At pH 6.0 in H2O, essentially one VO2+ ion remains bound to the protein with the metal site in the A conformation. Experiments with mixed VO2+ -Fe3+ transferrin complexes indicate that the same may be true of Fe3+. At pH 10.7, a new set of VO2+ resonances, labeled C, are observed; they possibly arise from a third conformation of the metal site. One bicarbonate or corbonate is required per VO2+ ion bound to the protein. 2.7 H+ are released per VO2+ bound in either the A or B conformations. The above results are discussed in terms of the "equivalence" and "nonequivalence" of the metal sites.     

Comparative study of the iron-binding properties of human transferrins: I. Complete and sequential iron saturation and desaturation of the lactotransferrin

Human lactotransferrin binds 2 Fe³⁺ tightly at two specific sites. In order to demonstrate differences between the stability of the two iron-binding sites, the removal of iron was studied in buffers in the pH range 8-3 varying the ionic strength and with or without metal chelators such as phosphate ions and EDTA.The results show that in the presence of formate and acetate buffers of ionic strength 0.1–0.4 and in a pH range of 5–3, the two Fe³⁺ from human lactotransferrin are removed stimultaneously.Addition of 4 mM EDTA to buffers of ionic strength 0.1 and in the pH range 8–3 shows that between pH 5–4.3 the iron from only one of the binding sites, called the ‘acid labile’ site, is removed.Addition of 0.2 M phosphate ions to buffers of ionic strength 0.2 and in pH range 8–3 containing 4 mM EDTA shows that Fe³⁺ from the ‘acid labile’ site may be completely removed at pH 6. Removal of Fe³⁺ from the ‘acid stable’ site is obtained at pH 4.The differential behavior of the two iron binding sites was also shown by saturation experiments in the presence of citrate/bicarbonate buffers at different pH values. In a pH range 6.2–4.8, 50% saturation was obtained, but at pH 6.35 complete saturation was achieved. When saturation of partially saturated samples of human lactotransferrin was performed with ⁵⁹Fe it was demonstrated that in the pH range 6.2–4.8 iron is bound only to the ‘acid labile’ site.     

Binding of vanadate to human serum transferrin

Human serum transferrin specifically and reversibly binds 2 equiv of vanadate at the two metal-binding sites of the protein. The vanadium(V)-transferrin complex can be formed either by the addition of vanadate to apotransferrin or by the air oxidation of the vanadyl(IV)-transferrin complex. The formation of the vanadium complex can be blocked by loading the apotransferrin with iron(III), and bound vanadium can be displaced from the protein by the subsequent addition of either gallium(III) or iron(III). The binding constant for the second equiv of vanadate is 10(6.5) in 0.1 M hepes, pH 7.4 at 25 degrees C. The binding constant for the first equiv of vanadate is probably very similar, although no quantitative value could be determined. Although transferrin reacts with the vanadate anion, studies on the transferrin model compound ethylenebis(o-hydroxyphenylglycine) indicate that at pH 9.5, the vanadium is binding at the metal-binding site as a dioxovanadium(V) cation coordinated to two phenolic residues at each binding site. This bound cation appears to be protonated over the pH range 9.5-6.5, as shown by changes in the difference uv spectrum of the transferrin complex, to produce an oxohydroxo species. Further decreases in the pH lead to dissociation of the vanadium-transferrin complex.     

Electron spin resonance of calmodulin-vanadyl complexes

X-band (9.2 GHz) electron spin resonance spectroscopy was used to investigate the binding of vanadyl to calmodulin. Solution spectra, obtained at ambient temperature with various VO2+:calmodulin molar ratios, suggested a binding stoichioimetry of 4 mol of VO2+/mol of protein and the possibility of two classes of binding sites. The latter was confirmed by using frozen solutions of calmodulin-VO2+ complexes that gave splitting of the spectral bands corresponding to the parallel components, which was particularly pronounced with the three high-field peaks. Competition of Ca2+ for the VO2+ binding sites was investigated, and the results indicated that two of the VO2+ sites corresponded to two of the Ca2+ sites; the other two VO2+ binding sites may have a higher affinity for VO2+ than for Ca2+ or they may correspond to Ca2+-independent sites. These results demonstrate that electron spin resonance spectroscopy can be used advantageously to probe subtle differences in the microenvironments of metal-binding sites in calmodulin.     

Spectrophotometric and ESR evidence for vanadium(IV) deferoxamine complexes.

Complexes of vanadium(IV), vanadyl, are reported to be formed with the trihydroxamic acid deferoxamine (H3DF+). One complex exhibits a reddish-violet color, with a major absorbance peak at 386 nm and a smaller peak at 520 nm. This complex is potentially useful for the microdetermination of vanadyl. The apparent molar absorptivity is 3.91 mM-1 cm-1, and the complex obeys Beer's law in the concentration range of 0.6-63 ppm. Electron spin resonance studies indicate the formation of two vanadyl complexes that are 1:1 in vanadyl and deferoxamine, but have two or three bound hydroxamate groups. ESR and spectrophotometric evidence indicate that the red, low pH form, involves an octahedral vanadium (4+) ion coordinated by three hydroxamate ligands. One of these hydroxamates is displaced by an oxygen at pH greater than 2.8 according to the following equilibria: VO2+ + H3DF+ in equilibrium with VIV(DF)2+ + H3O+, VIV(DF)2+ + H2O in equilibrium with VO(HDF)+ + H+, where pk2 = 2.8.     

Spectroscopic and thermodynamic studies on the binding of gadolinium(III) to human serum transferrin

A wide variety of thermodynamic, kinetic, and spectroscopic studies have demonstrated differences between the two metal-binding sites of transferrin. In the present investigation, we have further assessed these differences with respect to the binding of gadolinium, evaluated by UV difference spectrophotometry, electron paramagnetic resonance (EPR) titration, EPR difference spectroscopy in conjunction with urea gel electrophoresis, and equilibrium dialysis. Combinations of these studies establish that only one site of the protein binds Gd(III) sufficiently firmly to be characterized. In order to reveal which of the two sites accepts Gd(III), we made use of monoferric transferrins preferentially loaded with Fe(III) at either site in EPR spectroscopic studies. Because of the overlap of signals, difference spectroscopy was required to distinguish resonances arising from Fe(III) and Gd(III) specifically complexed to the protein. When iron is bound to the C-terminal site, leaving the N-terminal site free for binding of gadolinium, the difference spectrum shows no evidence of specific binding. However, when iron is bound to the N-terminal site, the difference spectrum shows a resonance line at g' = 4.1 indicative of specific binding, thus implicating the C-terminal site in the binding of Gd(III). The effective stability constant for the binding of Gd(III) to this site of transferrin at pH 7.4 and ambient pCO2 is 6.8 X 10(6) M-1. At physiological pCO2, the formation of nonbinding carbonato complexes of Gd(III) precludes a substantial role for transferrin in the transport of the lanthanide in vivo.     

Structure of the divalent metal ion activator binding site of S-adenosylmethionine synthetase studied by vanadyl(IV) electron paramagnetic resonance

The structure of the divalent metal ion binding site of S-adenosylmethionine synthetase from Escherichia coli has been studied by using the vanadyl(IV) ion (VO2+) as probe. VO2+ binds at a single site per subunit in the presence or absence of substrates. Single turnover experiments measuring S-adenosylmethionine (AdoMet) formation from methionine and the ATP analogue 5'-adenylyl imidodiphosphate show that complexes containing VO2+ and either Mg2+ or Ca2+ as a second metal ion are catalytically active, while a complex containing VO2+ alone is inactive. Electron paramagnetic resonance spectra of the enzyme-VO2+ complex, as well as complexes also containing AdoMet or methionine, indicate the coordination of two water molecules and at least two protein ligands to the VO2+. In complexes with polyphosphate substrates or products (e.g., enzyme-VO2+-ATP-methionine, enzyme-VO2+-PPi-Mg2+), EPR spectral changes reveal ligand substitutions on the VO2+, and 8.5-G isotropic superhyperfine coupling to two 31P nuclei can be resolved. 17O superhyperfine coupling from [17O]pyrophosphate indicates coordination of two oxygen atoms of PPi to the VO2+ ion. Thus the polyphosphate compounds are bidentate ligands to the VO2+, demonstrating that the VO2+ binds at the active site and suggesting a catalytic role for the protein-bound metal ion.     

Vanadyl(IV) conalbumin. I. Metal binding site configurations

Electron paramagnetic resonance (epr) and ultraviolet difference spectroscopy of vanadyl conalbumin indicate a binding capacity of two vanadyl ions, VO²⁺, per protein molecule in the pH 8–11 range; the binding capacity drops in the pH 6–8 range with an apparent pK_a′ = 6.6. Iron-saturated conalbumin does not bind vanadyl ions, which suggests common binding sites for iron and vanadium. Ultraviolet difference spectroscopy indicates 2–3 tyrosines are involved in the binding of each metal ion; pH titrations show that three protons are released per vanadyl ion bound by conalbumin. Room and liquid nitrogen temperature X-band (ca. 9.2–9.5 gHz) epr spectra show that the vanadyl ion binds in three magnetically distinct environments (A, B, and C) that arise from interconvertible metal site configurations. These configurations are probably examples of conformational substrates of the protein. Q-band (ca 34 gHz) epr spectra resolve the spectral features more clearly and show that two configurations (A and B) have axially symmetric epr parameters but angles of noncoincidence of 12° and 8°, respectively, between the z components of the g and nuclear hyperfine tensors. The third (C) configuration has rhombic magnetic symmetry and a 6° angle of noncoincidence. These observations demonstrate that the metal sites are of low symmetry and are flexible in their geometry about the metal.The isotropic g and nuclear hyperfine tensor values and the line widths used in computer-simulated epr spectra are consistent with four oxygen or three oxygen and one nitrogen donor atoms binding equatorially to the VO²⁺ group. The apparent stability constant indicates that vanadyl ion binds to conalbumin approximately twelve orders of magnitude more weakly than iron to human serotransferrin but still sufficiently strongly to overcome hydrolysis.     

Hydrophobic carriers of vanadyl ions augment the insulinomimetic actions of vanadyl ions in rat adipocytes.

A novel family of vanadyl ion (VO2+, oxidation state +4) carriers is introduced. These carriers possess C2 symmetry, utilize two hydroxamate groups as ion binding sites, and optionally possess asymmetric carbons. Binding efficiencies and hydrophobicities are regulated by the use of a modular assembly. When applied to rat adipocytes, these carriers augment the potency of vanadyl ions to stimulate glucose metabolism. The complexes shift the dose-response curve to the left. Also, the maximal effect of vanadyl ions which is in the order of 20-30% of that of insulin is shifted toward maximal (100-115%) stimulation. Among several chelators studied, the order of synergistic potency was RL-252 greater than or equal to RL-262 greater than 1367. RL-239, RL-280, and RL-261 had smaller effects, whereas RL-282 had a negligible effect. The synergistic action of RL-252 (and other chelators as well) on VO2+ was already observed at a molar ratio of 1:0.01 of VO2+ to RL-252, respectively, and maximal augmentation occurred at a molar ratio of 1:0.1. The superiority of the hydrophobic chelators relative to the hydrophilic ones, together with the low molar ratio of chelator to VO2+ to achieve maximal effect, strongly suggests that these chelators act as vanadyl ionophores. This notion was confirmed by carrier-facilitated extraction of VO2+ from water into CHCl3 with the following order of decreasing efficacy: RL-262 greater than RL-252 greater than 1367 greater than RL-261. The chelators' potentiating effect may therefore be related to facilitated transport of VO2+ ions into the cells' interiors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different metal-binding properties of the two sites of human transferrin.

Transferrin, the serum serum iron-transport protein which can bind two metal ions at physiologic pH, binds just one Fe3+, VO2+, or Cr3+ ion at pH 6.0. Fe3+ and VO2+ appear to be bound at the same site, designated A, based on electron paramagnetic resonance (EPR) spectra of VO2+-transferrin and (Fe3+)1(VO2+)1-transferrin. The EPR spectra of (Cr3+)1(VO2+)1-transferrin and of (Cr3+), (FE3+)1-transferrin indicate that that Cr3+ is bound to site B at pH 6.0. Transferrin was labeled at site A with 59Fe at pH 6.0 and at site B with 55Fe at pH 7.5. When the pH of the resulting preparation was lowered to 6.3 and the dissociated iron was separated by gel filtration, about ten times as much 55Fe as 59Fe was lost. The same EPR and isotopic-labeling experiments showed that Fe3+ added to transferrin at pH 7.5 binds to site A with about 90% selectivity.     

On the interaction of the vanadyl(IV) cation with lactose

A new vanadyl(IV) complex of the disaccharide lactose was obtained in aqueous solution at pH = 13. The sodium salt of the complex, of composition Na4[VO(lactose)2].3H2O, has been characterized by elemental analysis and by ultraviolet-visible, diffuse reflectance, and infrared spectroscopies. Its magnetic susceptibility and thermal behavior were also investigated. The inhibitory effect on alkaline phosphatase activity was tested for this compound as well as for the vanadyl(IV) complexes with maltose, sucrose, glucose, fructose, and galactose. For comparative purposes, the free ligands and the vanadyl(IV) cation were also studied. The free sugars and the sucrose/VO complex exhibited the lowest inhibitory effect. Lactose-VO, maltose-VO, and the free VO2+ cation showed an intermediate inhibition potential, whereas the monosaccharide/VO complexes appeared as the most potent inhibitory agents.     

Effect of vanadyl ions on calcineurin and its A subunit

Calcineurin (CN) exhibits a bimodal regulation by different concentrations of vanadyl ions (VO2+) in the presence of Mn2+. Low concentrations of VO2+ inhibit the enzyme, with 50 microM VO2+ completely inhibiting CN activity, while high concentrations, up to 500 microM VO2+, stimulate the CN activity. A similar bimodal regulation of CN was not observed with either calcium or vanadate under the same conditions. X-band electron spin resonance spectroscopy, used to study the binding of VO2+ to the catalytic subunit A of calcineurin, show that there are two kinds of binding sites in the A subunit.     

Binding patterns of vanadium ions with different valence states to human serum transferrin studied by HPLC/high-resolution ICP-MS

Vanadium (V) is an essential metal for mammals and has different valence states. In blood, V is bound to serum transferrin (Tf), a glycoprotein which has two metal-binding sites, and carbonate is generally required for the binding. In this study, the binding patterns of V(III), V(IV), and V(V) to human serum Tf (hTf) were analyzed using an HPLC system equipped with an anion-exchange column and directly connected to a high-resolution inductively coupled plasma-mass spectrometer for metal detection (51V). In affinity to hTf, the three ions were ranked V(III)>V(IV)>V(V) in the presence of bicarbonate and V(III) reverse congruent V(IV)>V(V) in the absence. Intermediates in the "open forms" binding to the respective sites were detected at the initial stage. V(IV) and V(V) were bound to the N-lobe site in the "closed form" and "open form," respectively. In the absence of bicarbonate, V ions with respective valence states were bound to hTf in the "open form." In terms of binding to hTf, tri-valent V was most favorable in the presence of bicarbonate.     

Steric restrictions on the binding of large metal ions to serum transferrin

Apotransferrin in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid at 25 degrees C and pH 7.4 was titrated with acidic solutions of Lu3+, Tb3+, and Eu3+. Metal binding at the two specific metal-binding sites of transferrin was followed from changes in the difference UV spectra at 245 nm. The binding of Tb3+ was also followed from changes in the fluorescence emission spectrum at 549 nm. Apotransferrin was titrated with solutions containing varying ratios of the metal ion and the competitive chelating agent nitrilotriacetic acid, and metal-transferrin binding constants were calculated by nonlinear least-squares fits of the absorbance as a function of titrant added. The sequential carbonate-independent equilibrium constants for the binding of two metal ions are log KM1 = 11.08 and log KM2 = 7.93 for Lu3+, log KM1 = 11.20 and log KM2 = 7.61 for Tb3+, and log KM1 = 9.66 and log KM2 = 7.27 for Eu3+. Titrations of both C-terminal and N-terminal monoferric transferrins indicate that all of these metal ions bind more strongly to the C-terminal binding site. The trend in log K values as a function of the lanthanide ionic radius has been evaluated both by plots of log K versus the metal ion charge/radius ratio and by linear free-energy relationships in which binding constants for complexes of the larger lanthanides are plotted versus the binding constants for complexes with the smallest lanthanide, Lu3+. Both methods indicate that there is a sharp drop in the binding constants for the C-terminal binding site for metals larger than Tb3+. This decrease is attributed to a steric hindrance to the binding of the larger cations. The steric effect is not as strong for metal binding at the N-terminal site. As a result, the selectivity for binding to the C-terminal site, which is quite high for the smaller lanthanides, drops sharply on going from Tb3+ to Nd3+.     

Structure of the manganese-bound manganese transport regulator of Bacillus subtilis

The Bacillus subtilis manganese transport regulator, MntR, binds Mn2+ as an effector and is a repressor of transporters that import manganese. A member of the diphtheria toxin repressor (DtxR) family of metalloregulatory proteins, MntR exhibits selectivity for Mn2+ over Fe2+. Replacement of a metal-binding residue, Asp8, with methionine (D8M) relaxes this specificity. We report here the X-ray crystal structures of wild-type MntR and the D8M mutant bound to manganese with 1.75 A and 1.61 A resolution, respectively. The 142-residue MntR homodimer has substantial structural similarity to the 226-residue DtxR but lacks the C-terminal SH3-like domain of DtxR. The metal-binding pockets of MntR and DtxR are substantially different. The cation-to-cation distance between the two manganese ions bound by MntR is 3.3 A, whereas that between the metal ions bound by DtxR is 9 A. D8M binds only a single Mn2+ per monomer, owing to alteration of the metal-binding site. The sole retained metal site adopts pseudo-hexacoordinate geometry rather than the pseudo-heptacoordinate geometry of the MntR metal sites.     

Interaction of divalent metal ions with the carboxyl-terminal domain of human voltage-gated proton channel Hv1

The voltage-gated proton channel Hv1 functions as a dimer, in which the intracellular C-terminal domain of the protein is responsible for the dimeric architecture and regulates proton permeability. Although it is well known that divalent metal ions have effect on the proton channel activity, the interaction of divalent metal ions with the channel in detail is not well elucidated. Herein, we investigated the interaction of divalent metal ions with the C-terminal domain of human Hv1 by CD spectra and fluorescence spectroscopy. The divalent metal ions binding induced an obvious conformational change at pH 7 and a pH-sensitive reduction of thermostability in the C-terminal domain. The interactions were further estimated by fluorescence spectroscopy experiments. There are at least two binding sites for divalent metal ions binding to the C-terminal domain of Hv1, either of which is close to His²⁴⁴ or His²⁶⁶ residue. The binding of Zn²⁺ to the two sites both enhanced the fluorescence of the protein at pH 7, whereas the binding of other divalent metal ions to the two sites all resulted fluorescence quenching. The orders of the strength of divalent metal ions binding to the two sites from strong to weak are both Co²⁺, Ca²⁺, Ni²⁺, Mg²⁺, and Mn²⁺. The strength of Ca²⁺, Co²⁺, Mg²⁺, Mn²⁺ and Ni²⁺ binding to the site close to His²⁴⁴ is stronger than that of these divalent metal ions binding to the site close to His²⁶⁶.     

Activation of rabbit liver high affinity cAMP (type IV) phosphodiesterase by a vanadyl-glutathione complex. Characterization of the role of the sulfhydryl.

Activation of rabbit liver microsomal high affinity cAMP phosphodiesterase (Type IV PDE) by vanadyl-glutathione complexes was studied as a possible model of insulin stimulation of the enzyme in a cell-free system. The effect of VO.2GSH activation of PDE was a 21-fold decrease in the IC50 value for cGMP inhibition and a 2.6-fold increase in the Vmax of the higher affinity cAMP catalytic site. Cyclic AMP and cGMP substrate affinities and cGMP hydrolysis were unaffected by VO.2GSH activation. Selective Type IV PDE inhibitors and cGMP analogs indicated that VO.2GSH complexes activated the cGMP-inhibitable form of the Type IV PDE activities which co-localized in hepatic microsomes. The Type IV PDE activating complex appears to consist minimally of vanadyl ion and 2 oxidized electron donor compounds. The components of the electron donor required to achieve an enzyme activation complex are: 1) a free -SH group as the electron donor for vanadate reduction and 2) a minimum structure of cysteamine (NH2-CH2-CH2-SH). Maximal activation of the enzyme required near 2:1 molar ratios of either glutathione or cysteamine mixed with sodium orthovanadate. Active vanadyl-cysteamine complexes were isolated by reverse- phase high performance liquid chromatography. Tungsten, niobium, and tantalum, but not manganese, chromium, or molybdenum, substituted for vanadium to form enzyme-activating complexes with glutathione. VO.RSH complex activation occurred rapidly upon addition to microsomes and was reversible. We conclude from these studies that VO.RSH complexes and insulin activate the same form of Type IV PDE in rabbit liver microsomes; our findings are discussed with respect to the involvement of a possible electron transfer enzyme oxidation in the activation mechanism.     

The influence of pH on the equilibrium distribution of iron between the metal-binding sites of human transferrin.

The dependence of the metal-binding properties of transferrin on pH in the pH 6--9 range was investigated by urea/polyacrylamide-gel electrophoresis. Equations are presented for calculating the relative values of the four conditional site constants for the stepwise binding of iron to the two sites of transferrin and for calculating the equilibrium distribution of the protein among the four principal forms, apotransferrin, the C-terminal and N-terminal monoferric transferrins and diferric transferrin. The relative affinity of iron for the two sites and the co-operativity of iron-binding follow characteristic "pH titration' curves. A mathematical model that can account for the former behaviour is presented. In both cases the metal-binding sites are affected by the ionization of functional groups with apparent pKa values near physiological pH approx. 7.4. There is strong positive co-operatively in the release of protons from these groups. The results indicate that pH must be accurately controlled in studies of the differential properties of the two sites of the transferrin molecule.