The pH-induced release of iron from transferrin investigated with a continuum electrostatic model.

A reduction in pH induces the release of iron from transferrin in a process that involves a conformational change in the protein from a closed to an open form. Experimental evidence suggests that there must be changes in the protonation states of certain, as yet not clearly identified, residues in the protein accompanying this conformational change. Such changes in protonation states of residues and the consequent changes in electrostatic interactions are assumed to play a large part in the mechanism of release of iron from transferrin. Using the x-ray crystal structures of human ferri- and apo-lactoferrin, we calculated the pKa values of the titratable residues in both the closed (iron-loaded) and open (iron-free) conformations with a continuum electrostatic model. With the knowledge of a residue's pKa value, its most probable protonation state at any specified pH may be determined. The preliminary results presented here are in good agreement with the experimental observation that the binding of ferric iron and the synergistic anion bicarbonate/carbonate results in the release of approximately three H+ ions. It is suggested that the release of these three H+ ions may be accounted for, in most part, by the deprotonation of the bicarbonate and residues Tyr-92, Lys-243, Lys-282, and Lys-285 together with the protonation of residues Asp-217 and Lys-277.     

Ferrous iron release from transferrin by human neutrophil-derived superoxide anion: effect of pH and iron saturation.

The ability of superoxide anion (O2-) from stimulated human neutrophils (PMNs) to release ferrous iron (Fe2+) from transferrin was assessed. At pH 7.4, unstimulated PMNs released minimal amounts of O2- and failed to facilitate the release of Fe2+ from holosaturated transferrin. In contrast, incubation of phorbol myristate acetate (PMA)-stimulated PMNs with holosaturated transferrin at pH 7.4 enhanced the release of Fe2+ from transferrin eightfold in association with marked generation of O2-. The release of Fe2+ was inhibited by addition of superoxide dismutase (SOD), indicating that the release of Fe2+ was dependent on PMN-derived extracellular O2-. In contrast, at physiologic pH (7.4), incubation of transferrin at physiological levels of iron saturation (e.g. 32%) with unstimulated or PMA stimulated PMNs failed to facilitate the release of Fe2+. The effect of decreasing the pH on the release of Fe2+ from transferrin by PMN-derived O2- was determined. Decreasing the pH greatly facilitated the release of Fe2+ from both holosaturated transferrin and from transferrin at physiological levels of iron saturation by PMN-derived O2-. Release of Fe2+ occurred despite a decrease in the amount of extracellular O2- generated by PMNs in an acidic environment. These results suggest that transferrin at physiologic levels of iron saturation may serve as a source of Fe2+ for biological reactions in disease states where activated phagocytes are present and there is a decrease in tissue pH. The unbound iron could participate in biological reactions including promoting propagation of lipid peroxidation reactions or hydroxyl radical formation following reaction with phagocytic cell-derived hydrogen peroxide.     

Mutation of the iron ligand His 249 to Glu in the N-lobe of human transferrin abolishes the dilysine "trigger" but does not significantly affect iron release

Serum transferrin is the major iron transport protein in humans. Its function depends on its ability to bind iron with very high affinity, yet to release this bound iron at the lower intracellular pH. Possible explanations for the release of iron from transferrin at low pH include protonation of a histidine ligand and the existence of a pH-sensitive "trigger" involving a hydrogen-bonded pair of lysines in the N-lobe of transferrin. We have determined the crystal structure of the His249Glu mutant of the N-lobe half-molecule of human transferrin and compared its iron-binding properties with those of the wild-type protein and other mutants. The crystal structure, determined at 2.4 A resolution (R-factor 19.8%, R(free) 29.4%), shows that Glu 249 is directly bound to iron, in place of the His ligand, and that a local movement of Lys 296 has broken the dilysine interaction. Despite the loss of this potentially pH-sensitive interaction, the H249E mutant is only slightly more acid-stable than wild-type and releases iron slightly faster. We conclude that the loss of the dilysine interaction does make the protein more acid stable but that this is counterbalanced by the replacement of a neutral ligand (His) by a negatively charged one (Glu), thus disrupting the electroneutrality of the binding site.     

Receptor-modulated iron release from transferrin: differential effects on N- and C-terminal sites

Iron release to PPi from N- and C-terminal monoferric transferrins and their complexes with transferrin receptor has been studied at pH 7.4 and 5.6 in 0.05 M HEPES or MES/0.1 M NaCl/0.01 M CHAPS at 25 degrees C. The two sites exhibit kinetic heterogeneity in releasing iron. The N-terminal form is slightly less labile than its C-terminal counterpart at pH 7.4, but much more facile in releasing iron at pH 5.6. At pH 7.4, iron removal by 0.05 M pyrophosphate from each form of monoferric transferrin complexed to the receptor is considerably slower than from the corresponding free monoferric transferrin. However, at pH 5.6, complexation of transferrin to its receptor affects the two forms differently. The rate of iron release to 0.005 M pyrophosphate by the N-terminal species is substantially the same whether transferrin is free or bound to the receptor. In contrast, the C-terminal form releases iron much faster when complexed to the receptor than when free. Urea/PAGE analysis of iron removal from free and receptor-complexed diferric transferrin at pH 5.6 reveals that its C-terminal site is also more labile in the complex, but its N-terminal site is more labile in free diferric transferrin. Thus, the newly discovered role of transferrin receptor in modulating iron release from transferrin predominantly involves the C-terminal site. This observation helps explain the prevalence of circulating N-terminal monoferric transferrin in the human circulation.     

Factors affecting the adenosine triphosphate induced release of iron from transferrin.

The release of iron from transferrin was investigated by incubating the diferric protein in the presence of potential iron-releasing agents. The effective chemical group appears to be pyrophosphate, which is present in blood cells as nucleoside di- and triphosphates, notably adenosine triphosphate (ATP). An alternative structure with comparable activity is represented by 2,3-diphosphoglycerate. Neither 1 mM adenosine monophosphate (AMP) nor 1 mM orthophosphate released iron from transferrin. The ATP-induced iron-releasing activity was dependent on weak acidic conditions and was sensitive to temperature and sodium chloride concentration. The rate of iron release rapidly increased as transferrin was titrated with HCl from pH 6.8 to 6.1 in the presence of 1 mM ATP and 160 mM NaCl at 20 degrees C. Iron release from transferrin without ATP was observed below pH 5.5. Ascorbate (10(-4) M) reduced Fe(III), but only after iron release from transferrin by a physiological concentration of ATP. A proposal for the mechanism of iron release from transferrin by ATP and the utilization of reduced iron by erythroid cells is described.     

Inhibition of reticulocyte iron uptake by NH4Cl and CH3NH2

The aim of this investigation was to test the hypothesis that elevation of intracellular pH would inhibit iron uptake by reticulocytes. The experiments were performed with rabbit reticulocytes and iron bound to rabbit transferrin. Incubation of the cells with NH₄Cl, (NH₄)₂CO₃, CH₃NH₂ and (CH₃)₂NH was used in an attempt to increase intracellular pH. These substances were all found to inhibit iron uptake by reticulocytes. The mechanism of action of NH₄Cl and CH₃NH₂ was investigated in detail. Similar results were found with both reagents. They inhibited iron uptake in a concentration-dependent manner, but produced a small increase in the cellular uptake of transferrin. The onset of action was rapid and the effect was reversible. There was no decrease in the number of transferrin-binding sites per cell and their apparent affinity for transferrin increased slightly, while the efficiency of iron removal from transferrin per binding site diminished greatly. The rate of transferrin release from reticulocytes was unaffected. NH₄Cl did not affect the rate of iron release from transferrin in a cell-free system. Incubation of reticulocytes with 10 mM NH₄Cl or CH₃NH₂ was found to produce an increase in intracellular pH of 0.05—0.15 pH units. The intracellular pH determined by used of the weak acid 5,5-dimethyl-oxazolidine-2,4-dione was significantly higher than that obtained with the weak base (CH₃)₂NH. By transmission electron microscopy it was shown that reticulocytes treated with NH₄Cl or CH₃NH₂ have enlarged intracellular vesicles. The results are considered to support the hypothesis that iron release from transferrin in reticulocytes occurs as a result of protonation of the transferrin within intracellular vesicles. According to this hypothesis, weak bases such as NH₃ and CH₃NH₂ inhibit iron release by neutralizing H⁺ within the vesicles.     

A new role for the transferrin receptor in the release of iron from transferrin

Iron removal by pyrophosphate from human serum diferric transferrin and the complex of transferrin with its receptor was studied in 0.05 M HEPES or MES buffers containing 0.1 M NaCl and 0.01 M CHAPS at 25 degrees C at pH 7.4, 6.4, and 5.6. At each pH, the concentration of pyrophosphate was adjusted to achieve rates of release amenable to study over a reasonable time course. Released iron was separated from protein-bound iron by poly(ethylene glycol) precipitation of aliquots drawn from the reaction mixture at various times during the course of a kinetic run. The amount of 59Fe label associated with the protein and pyrophosphate was determined from the radioactivity of precipitate and supernatant, respectively, in each aliquot. Iron removal of 0.05 M pyrophosphate at pH 7.4 from diferric transferrin bound to the receptor is considerably slower than that from free diferric transferrin, with observed pseudo-first-order rate constants of 0.020 and 0.191 min-1, respectively. For iron removal by 0.01 M pyrophosphate at pH 6.4, corresponding rate constants are 0.031 and 0.644 min-1. However, at pH 5.6, iron removal by 0.001 M pyrophosphate is faster from diferric transferrin bound to its receptor than from free transferrin (observed rate constants of 0.819 and 0.160 min-1, respectively). Thus, the transferrin receptor not only facilitates the removal of iron from diferric transferrin at the low pH that prevails in endocytic vesicles but may also reduce its accessibility to iron acceptors at extracellular pH, thereby minimizing the likelihood of nonspecific release of iron from transferrin at the cell surface.     

Human platelets mediate iron release from transferrin by adenine nucleotide-dependent and -independent mechanisms

We assessed the ability of platelet sonicates and mediators secreted by unstimulated and thrombin-stimulated platelets to facilitate the release of iron from transferrin. Platelet sonicates and platelet conditioned media potentiated the release of iron from transferrin. The rate of release of iron was dependent on the pH of the reaction and amount of platelet sample added. Conditioned media from thrombin-stimulated platelets was more effective in mediating the release of iron from transferrin than was conditioned media from unstimulated cells. The rate of iron released from transferrin following addition of ATP and ADP in amounts equivalent to that present in platelet conditioned media was significantly less than the rate of iron released following the addition of conditioned media from platelets. Depletion of ATP and ADP in platelet conditioned media by incubation with apyrase only partially inhibited their ability to enhance the rate of iron release from transferrin. These observations indicate that platelets enhance the release of iron from transferrin by adenine nucleotide-dependent and -independent mechanisms. These observations are consistent with the hypothesis that platelets promote oxidant-induced tissue injury at sights of inflammation secondary to their ability to enhance the local release of iron from transferrin.     

Intravesicular pH and iron uptake by immature erythroid cells

The intravesicular pH of intact rabbit reticulocytes was measured by two methods; one based on the intracellular:extracellular distribution of DMO (5, 5, dimethyl + oxazolidin-2,4-dione), methylamine, and chloroquine and the other by quantitative fluorescence microscopy of cell-bound transferrin. The latter method was also applied to nucleated erythroid cells from the fetal rat liver. A pH value of approximately 5.4 was obtained with both methods and in both types of cells. Treatment of the cells with lysosomotrophic agents, metabolic inhibitors, and ionophores elevated the intravesicular pH and inhibited iron uptake from transferrin. When varying concentrations of NH4Cl were used, a close correlation was observed between the inhibition of iron uptake and elevation of the intravesicular pH. At pH 5.4 iron release from rabbit iron-bicarbonate transferrin in vitro was much more rapid than from iron-oxalate transferrin. The bicarbonate complex donates its iron to rabbit reticulocytes approximately twice as quickly as the oxalate complex. It is concluded that the acidic conditions within the vesicles provide the mechanism for iron release from the transferrin molecule after its endocytosis and that the low vesicular pH is dependent on cellular metabolism.     

Tertiary structural changes associated with iron binding and release in hen serum transferrin: a crystallographic and spectroscopic study

The iron binding and release of serum transferrin are pH-dependent and accompanied by a conformational change between the iron-bound (holo-) and iron-free (apo-) forms. We have determined the crystal structure of apo-hen serum transferrin (hAST) at 3.5A resolution, which is the first reported structure to date of any full molecule of an apo-serum transferrin and studied its pH-dependent iron release by UV-vis absorption and near UV-CD spectroscopy. The crystal structure of hAST shows that both the lobes adopt an open conformation and the relative orientations of the domains are different from those of apo-human serum transferrin and human apolactoferrin but similar to that of hen apo-ovotransferrin. Spectroscopic analysis reveals that in hen serum transferrin, release of the first iron starts at a pH approximately 6.5 and continues over a broad pH range (6.5-5.2). The complete release of the iron, however, occurs at pH approximately 4.0. The near UV-CD spectra show alterations in the microenvironment of the aromatic residues surrounding the iron-binding sites.     

Iron exchange between transferrin molecules mediated by phosphate compounds and other cell metabolites.

The ability of a large number of cellular metabolites to release iron from transferrin was investigated by measuring the rate at which they could mediate iron exchange between two types of transferrin. Rabbit transferrin labelled with 59Fe was incubated with human apotransferrin in the presence of the metabolites. After varying periods of incubation the human transferrin was separated from the rabbit transferrin by immunoprecipitation. GTP, 2,3-diphosphoglycerate, ATP, ADP and citrate produced the most rapid exchange of iron between the two types of transferrin, but many other compounds showed some degree of activity. Iron exchange mediated by the organic phosphates had the characteristics of a single first-order reaction and was sensitive to changes of incubation temperature and pH. The activation energy for the exchange reaction was approx. 13 kcal/mol. The rate of iron exchange from the oxalate - iron - transferrin complex was much lower than from bicarbonate - iron - transferrin. It is concluded that several organic phosphates have the capacity of releasing iron from transferrin. These compounds may represent the means by which the iron is released during the process of cellular uptake.     

Dual role of Lys206-Lys296 interaction in human transferrin N-lobe: iron-release trigger and anion-binding site.

The unique structural feature of the dilysine (Lys206-Lys296) pair in the transferrin N-lobe (hTF/2N) has been postulated to serve a special function in the release of iron from the protein. These two lysines, which are located in opposite domains, hydrogen bond to each other in the iron-containing hTF/2N at neutral pH but are far apart in the apo-form of the protein. It has been proposed that charge repulsion resulting from the protonation of the dilysines at lower pH may be the trigger to open the cleft and facilitate iron release. The fact that the dilysine pair is positively charged and resides in a location close to the metal-binding center has also led to the suggestion that the dilysine pair is an anion-binding site for chelators. The present report provides comprehensive evidence to confirm that the dilysine pair plays this dual role in modulating release of iron. When either of the lysines is mutated to glutamate or glutamine or when both are mutated to glutamate, release of iron is much slower compared to the wild-type protein. This is due to the fact that the driving force for cleft opening is absent in the mutants or is converted to a lock-like interaction (in the case of the K206E and K296E mutants). Direct titration of the apo-proteins with anions as well as anion-dependent iron release studies show that the dilysine pair is part of an active anion-binding site which exists with the Lys296-Tyr188 interaction as a core. At this site, Lys296 serves as the primary anion-binding residue and Tyr188 is the main reporter for electronic spectral change, with smaller contributions from Lys206, Tyr85, and Tyr95. In iron-loaded hTF/2N, anion binding becomes invisible as monitored by UV-vis difference spectra since the spectral reporters Tyr188 and Tyr95 are bound to iron. Our data strongly support the hypothesis that the apo-hTF/2N exists in equilibrium between the open and closed conformations, because only in the closed form is Lys296 in direct contact with Tyr188. The current findings bring together observations, ideas, and experimental data from a large number of previous studies and shed further light on the detailed mechanism of iron release from the transferrin N-lobe. In iron-containing hTF/2N, Lys296 may still function as a target to introduce an anion (or a chelator) near to the iron-binding center. When the pH is lowered, the protonation of carbonate (synergistic anion for metal binding) and then the dilysine pair form the driving force to loosen the cleft, exposing iron; the nearby anion (or chelator) then binds to the iron and releases it from the protein.     

Iron uptake in reticulocytes. Inhibition mediated by the ionophores monensin and nigerisin

The lipophilic carboxylic ionophores monensin and nigerisin reversibly blocked iron uptake by erythroid cells. At low concentrations of ionophores (0.25-0.5 microM), the disruption of the compartment in which iron is released affected minimally the release of iron from transferrin but effectively inhibited iron uptake. Iron released from transferrin was extruded from the cell synchronously with but not bound to transferrin. The compartment disrupted by the ionophores, and in which iron is released from transferrin, is apparently contiguous to the extracellular medium. Contiguity was assessed by determining the effect of extracellular Na+ and K+ on the activity of the ionophores. The above data fit a model of iron uptake in which iron is released from transferrin in an acidic compartment in immediate contiguity with the cell plasma membrane. Iron is then bound by its membrane acceptor and is translocated to the cytosolic side of the plasma membrane. At submicromolar concentrations, the ionophores monensin and nigerisin produce a small increase in the pH of the acidic compartment. The pH change, which is not sufficient to block the release of iron from transferrin, is enough to block the binding of released iron to its acceptor in the plasma membrane, thus producing inhibition of iron uptake.     

Iron exchange between transferrin molecules mediated by phosphate compounds and other cell metabolites

The ability of a large number of cellular metabolites to release iron from transferrin was investigated by measuring the rate at which they could mediate iron exchange between two types of transferrin. Rabbit transferrin labelled with ⁵⁹Fe was incubated with human apotransferrin in the presence of the metabolites. After varying periods of incubation the human transferrin was separated from the rabbit transferrin by immunoprecipitation.GTP, 2,3-diphosphoglycerate, ATP, ADP and citrate produced the most rapid exchange of iron between the two types of transferrin, but many other compounds showed some degree of activity. Iron exchange mediated by the organic phosphates had the characteristics of a single first-order reaction and was sensitive to changes of incubation temperature and pH. The activation energy for the exchange reaction was approx. 13 kcal/mol. The rate of iron exchange from the oxalate · iron · transferrin complex was much lower than from bicarbonate · iron · transferrin.It is concluded that several organic phosphates have the capacity of releasing iron from transferrin. These compounds may represent the means by which the iron is released during the process of cellular uptake.     

The preparation, properties, and metabolism of 14C-bicarbonate-labelled transferrin

¹⁴C-bicarbonate-labelled transferrin was prepared in order to study the role of bicarbonate in the cell-mediated release of iron from transferrin. ¹⁴C-bicarbonate bound to transferrin only in the presence of iron and with a ratio of bound bicarbonate to bound iron of one. The transferrin-¹⁴C-bicarbonate complex was very stable in Tris-HCl buffered at pH 7.5–9.0 even in the presence of excess non-radioactive bicarbonate. However, oxalate, citrate, and phosphate promoted a rapid exchange of transferrin-bound ¹⁴C-bicarbonate with bicarbonate present in the medium.Rabbit reticulocytes effected a temperature-dependent release of ¹⁴C-bicarbonate from transferrin at the same rate at which they incorporated ⁵⁹Fe from transferrin — suggesting the existence of a coordinated mechanism in the cells for the release of both iron and bicarbonate from transferrin.     

Receptor-induced switch in site-site cooperativity during iron release by transferrin.

Iron removal by PPi from the N- and C-terminal binding sites of both free and receptor-complexed transferrin, when the partner site remains occupied with kinetically inert Co(III), has been studied at pH 7.4 and 5.6, at 25 degrees C. At extracellular pH, 7.4, the C-terminal site of free mixed-metal proteins is slightly more labile than its N-terminal counterpart in releasing iron to 0.05 M PPi. The rate and extent of iron removal are retarded from both sites when transferrins are receptor-bound. At endosomal pH, 5.6, the two sites exhibit greater kinetic heterogeneity in iron release to 0.005 M PPi. The N-terminal site is 6 times more facile in relinquishing iron than the C-terminal site when mixed-metal transferrins are free. However, the two sites are affected oppositely upon binding to the receptor. Iron release from the C-terminal site of receptor-complexed CoN-transferrin-FeC is 4 times faster than that from receptor-free protein. In contrast, iron removal from the N-terminal site of receptor-complexed FeN-transferrin-CoC is slowed by a factor of 2 compared to that from free protein. These results help explain our previous observation of a receptor-induced switch in site lability during iron removal from diferric transferrin at pH 5.6 (Bali & Aisen, 1991). Site-site cooperative interactions between the two sites of doubly-occupied transferrin during iron release are altered upon binding to receptor at pH 5.6. Iron in the otherwise weaker binding site of the N-terminal lobe is stabilized, while iron in the relatively stable binding site of the C-terminal lobe is labilized.     

Iron release from transferrin, its C-lobe, and their complexes with transferrin receptor: presence of N-lobe accelerates release from C-lobe at endosomal pH

Human transferrin, like other members of the transferrin class of iron-binding proteins, is a bilobal structure, the product of duplication and fusion of an ancestral gene during the course of biochemical evolution. Although the two lobes exhibit 45% sequence identity and identical ligand structures of their iron-binding sites (one in each lobe), they differ in their iron-binding properties and their responsiveness to complex formation with the transferrin receptor. A variety of interlobe interactions modulating these iron-binding functions has been described. We have now studied the kinetics of iron release to pyrophosphate from the isolated recombinant C-lobe and from that lobe in the intact protein, each free and bound to receptor. The striking finding is that the rates of iron release at the pH of the endosome to which transferrin is internalized by the iron-dependent cell are similar in the free proteins but 18 times faster from full-length monoferric transferrin selectively loaded with iron in the C-lobe than from isolated C-lobe when each is complexed to the receptor. The possibility that the faster release in the receptor complex of the full-length protein at endosomal pH contributes to the evolutionary advantage of the bilobal structure is considered.     

The effect of pH on the kinetics of iron release from human transferrin

The rate of iron release from the N-terminal and C-terminal monoferrictransferrins (Fe_N-transferrin and Fe_C-transferrin, respectively) has been studied at 37°C over the pH range 3.5–10.6 using EDTA as the accepting chelate. Fe_N-transferrin is the more facile except above pH 8.2. Plots of log₁₀k_obs against pH showed a deviation for both monoferrictransferrins between pH 5.6 and 6.0 and studies above and below this transition point indicated that iron release occures by different mechanisms. At low pH (< 5.6) the rate of release from Fe_N-transferrin is independent of the presence of EDTA or NaClO₄, whereas Fe_c-transferrin shows a small but significant increase with increasing EDTA concentration. Rapid protonation of both monoferrictransferrins is followed by relatively slow release of Fe³⁺ which is subsequently chelated by EDTA. The slower release from Fe_c-transferrin is probably due to its greater binding strength for iron and the greater conformational stability of the C-terminal domain. Above pH 6.0 iron release from both monoferrictransferrins increases as the concentration of EDTA is increased. Direct attacks of EDTA probably occurs giving Fe-transferrin (HCO₃). EDTA as a transition state or intermediate. The factors which may lead to the observed pH dependence of the rate include (i) protonation of groups directly bound to the iron, (ii) conformers which differ in degree of protonation and (iii) the degree of protonation of the attacking chelating agent. It is suggested that an increase in conformational fluctuations as the pH is lowered may play a very important role. Studies with differrictransferrin at pH 4.53 and 7.40 showed that when iron is released to EDTA the rate is independent of the occupancy of the other site; that is, the two sites are exhibiting non-co-operativity.     

The effect of lysosomotrophic bases and inhibitors of transglutaminase on iron uptake by immature erythroid cells

The mechanism by which weak bases block iron uptake by immature erythroid cells was investigated using rabbit and rat reticulocytes and erythroblasts from the fetal rat liver. A large variety of bases was found to inhibit iron uptake but to have a much smaller or no effect on transferrin uptake by the cells. Quinacrine and chloroquine were active at the lowest concentrations. Dansylcadaverine, an inhibitor of transglutaminase, was also active at low concentration. However, the results do not indicate a role for transglutaminase in the iron uptake process. Instead they show that the major effect of the bases is to inhibit iron release from transferrin molecules on or within the cells. The possible mechanism of this effect was investigated by measurement of intracellular ATP levels, intracellular pH and by morphological studies utilizing fluorescent and electron microscopy. The bases caused little change in ATP levels, but elevated intracellular pH, probably due to accumulation within intracellular vesicles, which were shown to accumulate fluorescent weak bases, to swell under the action of the bases and to be the site of intracellular localization of transferrin. It is concluded that the bases tested in this work inhibit iron release from transferrin in intracellular vesicles by increasing their pH rather than by blocking transglutaminase and thereby restricting endocytosis. Reduction of transferrin uptake by the cells when it occurs is probably due to inhibition of recycling of transferrin receptors to the outer cell membrane.     

Studies on the mechanism of iron release from transferrin.

Iron release from human, rabbit, rat and sheep transferrin, chicken conalbumin and human lactoferrin was measured by the change in absorbance of solutions of the iron-protein complexes or by the release of 59Fe from the protein conjugated to agarose. Several phosphatic compounds and iron chelators were able to mediate the process (ATP, GTP, 2,3-diphosphoglycerate, inositol hexaphosphate, pyridoxal 5-phosphate, cytidine 5-triphosphate, pyrophosphate, inorganic phosphate, citrate, EDTA, oxalate, nitrilotriacetate). The greatest rate of iron release was found with pyrophosphate and the least with inorganic phosphate. Different rates of iron release were obtained with the different proteins, greatest with human transferrin and least with lactoferrin. With each of the proteins and the mediators there was a linera relationship between the H+ concentration and the rate of iron release. At any given pH the rate of iron release increased to a maximal rate as the mediator concentration was raised. It is concluded that iron release from transferrin under the conditions of these experiments involves an initial interaction between H+ and the iron-transferrin complex followed by release of the iron under the action of the mediator.