Monoferric Transferrin Research Articles

Binding of phosphonate chelating agents and pyrophosphate to apotransferrin.

Difference ultraviolet spectroscopy has been used to monitor the binding of a series of phosphonate ligands to human apotransferrin. The ligands consist of pyrophosphate as well as the phosphonic acids (aminomethyl)phosphonic acid (AMPA), (hydroxymethyl)phosphonic acid (HMP), (phosphonomethyl)-iminodiacetic acid (PIDA), N,N-bis(phosphonomethyl)glycine (DPG), and nitrilotris(methylenephosphonic acid) (NTP). Equilibrium constants have been measured for the sequential binding of two ligands per molecule of apotransferrin. In addition, site-specific equilibrium constants have been measured for the binding of AMPA, HMP, and PIDA to the vacant binding site of both forms of monoferric transferrin. Since titrations of diferric transferrin produce no difference UV spectrum, it is proposed that the primary binding site for phosphonic acids includes the protein groups that bind the synergistic bicarbonate anion that is required for formation of a stable ferric transferrin complex. It is further proposed that those ligands with two phosphonate groups can simultaneously bind to cationic amino acid side chains that extend into the cleft between the two domains of each lobe of transferrin. From an inspection of the ferric transferrin crystal structure, the most likely anion binding residues in the cleft are Arg-632 and Lys-534 in the C-terminal lobe and Lys-206 and Lys-296 in the N-terminal lobe.

Kinetics of iron removal from monoferric and cobalt-labeled monoferric human serum transferrin by nitrilotris(methylenephosphonic acid) and nitrilotriacetic acid

The kinetics of iron removal from both forms of monoferric transferrin by nitrilotris(methylenephosphonic acid) (NTP) and nitrilotriacetic acid (NTA) have been studied in 0.1 M, pH 7.4 N-(2-hydroxyethyl)piperazine-N'-ethanesulfonate buffer at 25°C. The dependence of the observed pseudo-first-order rate constant for removal of iron on the concentration of NTP has been interpreted in terms of two parallel pathways: one that saturates and another that is first order in ligand. For NTP the saturation pathway is more important, while the carboxylate analogue NTA removes iron from both sides entirely by a first-order process

Iron and the Liver

Iron is essential for life, but iron overload is toxic and potentially fatal. The liver is a major site of iron storage and is particularly susceptible to injury from iron overload, especially when (as in primary hemochromatosis) the iron accumulates in hepatocytes. Iron can be taken up by the liver in several forms and by several pathways including: (1) receptor-mediated endocytosis of diferric or monoferric transferrin or ferritin, (2) reduction and carrier-facilitated internalization of iron from transferrin without internalization of the protein moiety of transferrin, (3) electrogenic uptake of low molecular weight, non-protein bound forms of iron, and (4) uptake of heme from heme-albumin, heme-hemopexin, or hemoglobin-haptoglobin complexes. Normally, pathway 2 is probably the major one for uptake of iron by hepatocytes. Iron is stored in the liver in the cores of ferritin shells and as hemosiderin, an insoluble product derived from iron-rich ferritin. Iron in hepatocytes stimulates translation of ferritin mRNA and represses transcription of DNA for transferrin and transferrin receptors. The major pathologic effects of chronic hepatic iron overload are: (1) fibrosis and cirrhosis, (2) porphyria cutanea tarda, and (3) hepatocellular carcinoma. Although precise pathogenetic mechanisms remain unknown, iron probably produces these and other toxic effects by increasing oxidative stress and lysosomal lability. Vigorous efforts at diagnosis and treatment of iron overload are essential since the pathologic effects of iron are totally preventable by early vigorous iron removal and prevention of iron re-accumulation.

Metal ligand-induced alterations in the surface structures of lactoferrin and transferrin probed by interaction with immobilized copper(II) ions

We have evaluated immobilized Cu(II) ions as a potential site-directed molecular probe to monitor ligand-induced alterations in protein surface structures. Metal ion-induced alterations in the surface structures of different lactoferrins (human and porcine), transferrins (human and rabbit), and ovotransferrin (chicken) were examined. Although these 78 000-dalton glycoproteins are related gene products with similar overall structure and function, they differ greatly in the number and distribution of surface-exposed electron-donor groups thought to interact with Cu(II) ions. Each of these proteins interacted with immobilized Cu(II) ions through sites which are distinct from the two specific high affinity metal binding sites identified for iron. In both the presence and absence of bound iron, transferrins interacted more strongly with the immobilized Cu(II) ions than did lactoferrins; ovotransferrin interacted only weakly. Although iron binding increased the affinities of lactoferrins for immobilized Cu(II), iron binding decreased the affinities of transferrins and ovotransferrin for immobilized Cu(II) ions. Iron-saturated and iron-free lactoferrins were resolved by pH gradient elution, but only in the presence of 3 M urea; they were not resolved by imidazole affinity elution. Conversely, the iron-saturated and iron-free forms of transferrin were only separated by imidazole affinity elution. Urea did not influence the resolution of apo and holo ovotransferrins by imidazole. The differential effects of urea and imidazole suggest the participation of different types of surface electron-donor groups. The progressive site-specific modification of surface-exposed histidyl residues by carboxyethylation revealed several lactoferrin forms of intermediate affinity for immobilized iminodiacetate—Cu(II) ions. In summary, independent of species, the affinity for immobilized Cu(II) ions increased as follows: iron-saturated ovotransferrin < metal-free ovotransferrin < apolactoferrin < hololactoferrin << diferric or holotransferrin < monoferric transferrin < apotransferrin. We have demonstrated the use of immobilized Cu(II) ions to distinguish and to monitor ligand-induced alterations in protein surface structure. The results are discussed in relation to protein surface-exposed areas of electron-donor groups.

Site-specific rate constants for iron removal from diferric transferrin by nitrilotris(methylenephosphonic acid) and pyrophosphate

The kinetics of iron removal from human serum diferric transferrin by nitrilotris(methylenephosphonic acid) (NTP) and pyrophosphate ( PP i) have been studied in 0.1 m, pH 7.4, N-(2-hydroxyethyl)piperazine- N′-2-ethanesulfonate buffer at 25 °C using urea/polyacrylamide gel electrophoresis. The four microscopic rate constants required for a complete description of iron removal from the two transferrin metal-binding sites have been measured at 100 m m concentrations of NTP and PP i. There is very good agreement between the rate constants determined by electrophoresis in this study and the corresponding rate constants determined spectrophotometrically for monoferric transferrins that have been labeled at the empty binding site with substitutionally inert Co(III). The results validate the use of cobalt-labeled transferrins as models for kinetic studies on iron removal from diferric transferrin.

The effects of anions on the kinetics of reductive elimination of iron from monoferrictransferrins by thiols

The kinetics of reductive elimination of iron from the human serum monoferric transferrins by thioglycollate (TG), 3-mercaptopropionate (MP), cysteine (Cys), cysteamine (Cym) and 2-mercaptoethanol (ME) have been studied at 37°C using bathophenanthroline sulphonate (BPS) as the ferrous ion acceptor. Analysis of the entire course of the reaction was possible only with thioglycollate since the other thiols cause eventual protein precipitation; in these cases, initial rates were used. The rate of iron release is linearly dependent on thiol concentration at low concentrations of reductant (<approx. 0.2 M) and increases more rapidly with higher concentrations (up to 0.5–0.75 M). The thiols fall into two distinct groups, with TG, MP, and Cys reacting at approx. the same rate, which is an order of magnitude faster than the reaction with Cym and ME. The carboxylate functionality present in the first group may be responsible for the faster reaction rate, by competitively weakening the interaction between the protein and synergistic anion. The pH-dependence of the rate of reductive elimination appears to depend on ionizable functionalities on both the protein and reducing agent. The addition of NaCl, NaClO 4, NaHCO 3 and Na 2HPO 4 increases the rate of iron release from the monoferric transferrins. The last two have particularly large accelerating effects and, in the case of the N-terminal monoferrictransferrin, gave saturation kinetics, suggesting that the observed effect is due to conformational changes in the protein caused by binding of ions. The role of the Fe-synergistic anion complex in the transferrins as a ‘trapped intermediate’ is considered.

Occupancy of the iron-binding sites of human transferrin in sera obtained from different anatomical sites.

Transferrin is the major iron-transport protein in serum. Four molecular species (apo-, two mono-, and diferric transferrins) can be distinguished on the basis of their occupancy with iron. These species differ physicochemically and in the affinity with which they bind to the transferrin receptor. To elucidate the possible role of the four molecular species in directing the flow of iron between the major anatomical sites of iron release and utilization we have analyzed sera from eight stable multiorgan donors. The samples were obtained from veins draining the spleen, gut, and liver, and from the periphery. Employing polyacrylamide-gel electrophoresis in combination with crossed immunoelectrophoresis we were able to identify the four molecular species in all samples. Apo-transferrin was the predominant molecular species while diferric transferrin was the least abundant (P less than 0.01). The monoferric species were dominated by the acid-stable form (iron loading on the C-terminal end of the molecule) with ratios C/N from 1.2 (splenic and posthepatic serum) to 1.5-1.6 in mesenteric or peripheral samples respectively. We conclude from our study that it is unlikely that the monoferric transferrin species play a role in directing internal iron exchange and that in accordance with most of the literature the acid-stable form is preferentially loaded under physiological conditions of iron metabolism.

Iron release from transferrin induced by mixed ligand complexes of copper(II)

Copper(II) complexes CuL1L2 with the ligand pairs 3-phosphoglycerate (PG)/ethylenediamine (en), phosphoserine (PS)/ethylenediamine, phosphoserine/malonate (mal) are shown to be effective in inducing the release of both iron atoms from di-ferric transferrin (Fe2Tf; human serum transferrin) at pH 7.3 in 1 M NaCl at 25 degrees C. Half-times of the reaction with Cu(PG)(en)- were less than 1 min at 0.02 M concentration. The iron(III) products are polynuclear hydroxo complexes. There is weaker interaction with Cu(PS)4-2 and virtually none with Cu(serine)(en) nor Cu(PS)(2,2'-bipyridyl)-, revealing crucial effects of the combined ligand sphere including the phosphomonoester group. The results suggest that the release of iron from Fe2Tf, or from either monoferric transferrins, occurred due to the breakdown of the stability of iron binding in conjunction with the expulsion of the synergistic anion carbonate (or oxalate). The active copper(II) complexes are postulated to be models of membrane components that could liberate iron from transferrin succeeding its uptake at the receptor sites of cells.

Further evaluation of the biphasic kinetics of iron removal from transferrin by 3,4-LICAMS.

Further evaluation of the kinetic data for Fe3+ removal from isolated differic and monoferric transferrins by the tricatechol ligand 3,4-LICAMS has allowed full characterization of the four microscopic rate constants. A very small cooperativity exists between the two iron-binding sites with respect to their rates of iron release. The activation free energy profile for the system is presented.

The bicarbonate-dependence of zinc(II)-transferrin binding

The binding of zinc(II) to human serum transferrin has been studied as a function of the solution concentration of sodium bicarbonate in 100 mM, pH 7.4 hepes buffer at 25°C. The apparent molar absorptivity of the zinc-transferrin complex has been determined from the initial slopes of titration curves of Δϵ versus the ratio of [Zn] [Tf] . This absorptivity represents the difference between the positive absorbance of the ternary Zn-HCO 3-Tf species in the sample cuvette and the negative absorbance of binary HCO 3-Tf species in the reference cuvette. Higher concentrations of bicarbonate increase the degree of saturation of apo-Tf with bicarbonate and thus increase the apparent absorptivity of the zinc-Tf complex. Titrations of apo- and monoferric transferrins with bicarbonate indicate that there is little, if any, difference in the bicarbonate binding constants of the two specific transferrin binding sites. An equilibrium constant of log K = 2.49 has been used to calculate the degree of saturation of the C-terminal binding site with bicarbonate. The zinc-binding affinity of this site depends linearly on this degree of saturation. The scatter in the zinc-binding constants of the weaker N-terminal site precludes a similar analysis of the bicarbonate-dependence of binding at this site. The results strongly support the previous proposal that binding of the synergistic bicarbonate anion is responsible for the uv absorption observed upon addition of bicarbonate to apoTf.

Random distribution of iron among the two binding sites of transferrin in patients with various hematologic disorders

The distribution of iron among the two binding sites of transferrin was studied by isoelectric focusing in 41 patients with a variety of disorders of iron metabolism and erythropoiesis. The proportion of diferric and the two monoferric transferrins were very close to the values predicted from the transferring saturation. It is concluded that iron distribution on transferrin is random or close to random within the experimental error in patients with a variety of clinical disorders.

Effects of ionic strength on iron removal from the monoferric transferrins

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTEffects of ionic strength on iron removal from the monoferric transferrinsSuzanne A. Kretchmar and Kenneth N. RaymondCite this: Inorg. Chem. 1988, 27, 8, 1436–1441Publication Date (Print):April 1, 1988Publication History Published online1 May 2002Published inissue 1 April 1988https://doi.org/10.1021/ic00281a028RIGHTS & PERMISSIONSArticle Views123Altmetric-Citations49LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (546 KB) Get e-Alerts Get e-Alerts

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.

Nonrandom distribution of iron in circulating human transferrin

By combining the urea gel electrophoresis technique of Makey and Seal with Western immunoblotting, a method has been developed for analyzing the distribution of iron between the two sites of circulating human transferrin. The new method avoids exposure of samples to a nonphysiologic pH that may promote removal or redistribution of iron from the protein; this facilitates examination of multiple samples at one time. Analysis of 21 freshly drawn specimens from normal human subjects confirms previous reports that iron is not randomly distributed in the specific sites of transferrin. Rather, there is a considerable range in the ratio of occupancies of N-terminal and C- terminal sites (N:C ratio), from 0.31 to 6.87 in the present study, with the N-terminal site predominantly occupied in most subjects. The N:C ratio correlates modestly with serum iron concentration (r = .54). Possible flaws in studies indicating a random occupancy of the specific sites of circulating transferrin may lie in the low pH to which samples may be exposed during procedures based on isoelectric focusing or in drawing inferences from data considering only total monoferric transferrin rather than the two distinguishable monoferric species.

Nonrandom Distribution of Iron in Circulating Human Transferrin

By combining the urea gel electrophoresis technique of Makey and Seal with Western immunoblotting, a method has been developed for analyzing the distribution of iron between the two sites of circulating human transferrin. The new method avoids exposure of samples to a nonphysiologic pH that may promote removal or redistribution of iron from the protein; this facilitates examination of multiple samples at one time. Analysis of 21 freshly drawn specimens from normal human subjects confirms previous reports that iron is not randomly distributed in the specific sites of transferrin. Rather, there is a considerable range in the ratio of occupancies of N-terminal and C-terminal sites (N:C ratio), from 0.31 to 6.87 in the present study, with the N-terminal site predominantly occupied in most subjects. The N:C ratio correlates modestly with serum iron concentration (r = .54). Possible flaws in studies indicating a random occupancy of the specific sites of circulating transferrin may lie in the low pH to which samples may be exposed during procedures based on isoelectric focusing or in drawing inferences from data considering only total monoferric transferrin rather than the two distinguishable monoferric species.

Fluorescence probe measurement of the pH of the transferrin microenvironment during iron uptake by rat bone marrow erythroid cells.

Fluorescence probe measurements of the transferrin micro-environment during iron uptake by rat erythroid cells revealed that part of the transferrin is taken up in an acidic environment. The pH of this intracellular transferrin environment is 5.7. When rat erythroid cell precursors are incubated with diferric transferrin then in the incubation medium monoferric transferrins TfNFe and TfFeC appear. In view of the known instability of TfNFe at acidic pH, TfNFe cannot arise after endocytosis of Tf2Fe in acid vesicles at pH below 6.0. The results support the existence of a mechanism other than endocytosis in the iron uptake process in rat erythroid cells.

Quantitation of Apo-, Mono-, and Diferric Transferrin by Polyacrylamide Gradient Gel Electrophoresis in Patients With Disorders of Iron Metabolism

We have developed a polyacrylamide gradient gel electrophoretic method to quantitate apo-, mono-, and diferric transferrin based upon differences in their molecular size. Purified transferrin saturated to different extents (3% to 98%) with iron showed proportions of the three forms as predicted from an approximately random distribution of iron between the two metal-binding sites. The iron distributions in sera of 14 normal individuals similarly correlated with the predicted values. In contrast, 22 of 43 patients with diseases associated with abnormalities in iron or transferrin metabolism had a disproportionate increase in monoferric transferrin. This abnormality occurred in seven of nine patients who had received bone marrow transplants, seven of 14 with chronic liver disease, and eight of nine menstruating women with probable iron deficiency anemia. Interestingly, 11 patients with malabsorption or chronic renal disease had normal iron distributions. The finding of abnormal distributions of iron on transferrin suggests that gradient gel analysis may be a useful tool for studying the physiologic mechanisms controlling iron utilization.

Quantitation of apo-, mono-, and diferric transferrin by polyacrylamide gradient gel electrophoresis in patients with disorders of iron metabolism

We have developed a polyacrylamide gradient gel electrophoretic method to quantitate apo-, mono-, and diferric transferrin based upon differences in their molecular size. Purified transferrin saturated to different extents (3% to 98%) with iron showed proportions of the three forms as predicted from an approximately random distribution of iron between the two metal-binding sites. The iron distributions in sera of 14 normal individuals similarly correlated with the predicted values. In contrast, 22 of 43 patients with diseases associated with abnormalities in iron or transferrin metabolism had a disproportionate increase in monoferric transferrin. This abnormality occurred in seven of nine patients who had received bone marrow transplants, seven of 14 with chronic liver disease, and eight of nine menstruating women with probable iron deficiency anemia. Interestingly, 11 patients with malabsorption or chronic renal disease had normal iron distributions. The finding of abnormal distributions of iron on transferrin suggests that gradient gel analysis may be a useful tool for studying the physiologic mechanisms controlling iron utilization.

The effect of iron binding on the conformation of transferrin. A small angle x-ray scattering study

Distance distribution functions, p(r), radii of gyration, Rg, and radii of gyration of cross section, Rq, of apotransferrin, monoferric transferrin, and diferric transferrin have been compared. The alteration of Rg and Rq upon iron binding has been determined by a difference method. An unusual feature of the stepwise structural changes of transferrin upon iron saturation is that binding of the first ferric ion is responsible for more than half of the whole change in Rq, whereas Rg alters significantly only after the binding of the second ferric ion.

Transferrin saturation, plasma iron turnover, and transferrin uptake in normal humans

The relationship between plasma iron, transferrin saturation, and plasma iron turnover was studied in 53 normal subjects whose transferrin saturation varied between 17% and 57%, in 25 normal subjects whose transferrin saturation was increased by iron infusion to between 67% and 100%, and in five subjects with early untreated idiopathic hemochromatosis whose transferrin saturation was continually elevated to between 61% and 86%. The plasma iron turnover of all of these subjects ranged from 0.45 to 1.22 mg/dL whole blood/d. The mean values for the above-mentioned three groups were 0.71 +/- 0.17, 1.01 +/- 0.11, and 1.01 +/- 0.13 mg/dL whole blood/d, respectively. Most of this variation, estimated at 72% by regression analysis, was due to a direct relationship between transferrin saturation and plasma iron turnover. This effect was attributed to a competitive advantage of diferric over monoferric transferrin in delivering iron to tissues. This was confirmed by the demonstration of a more rapid clearance of diferric as compared to monoferric transferrin in an additional group of eight normal subjects. Calculations were made of the amount of transferrin reacting with membrane receptors per unit time. Allowance was made for the noncellular (extravascular) exchange and for the 4.2:1 preference of diferric over monoferric transferrin demonstrated in vitro. The amount of iron-bearing transferrin leaving the plasma to bind to tissue receptors for 53 subjects with a transferrin saturation between 17% and 57% was 71 +/- 13; for 25 subjects with a saturation from 67% to 100%, 72 +/- 12; and for five subjects with early idiopathic hemochromatosis, 82 +/- 11 mumol/L whole blood/d. There were no significant differences among these groups. These studies indicate that while the number of iron atoms delivered to the tissues increases with increasing plasma iron and transferrin saturation, the number of iron-bearing transferrin molecules that leave the plasma per unit time to bind to tissue receptors is relatively constant and within the limits studied, independent of transferrin saturation.