Sites Of Transferrin Research Articles

Distribution of iron between the binding sites of transferrin in serum: methods and results in normal human subjects

When it is incompletely saturated with iron, transferrin may exist in four molecular forms: apotransferrin, monoferric (A) transferrin (with iron occupying only the A site of the protein), monoferric (B) transferrin, and diferric transferrin. By combining electrophoresis in urea-polyacrylamide gels with crossed immunoelectrophoresis using specific antihuman transferin antiserum, it is possible to display and estimate the concentration of each of these four forms in normal human serum. The distribution of iron between the binding sites of transferrin is neither random nor determined by the relative binding strengths of transferrin's two sites. Rather, the more weakly binding and acid-labile B site of the protein is predominantly occupied.

Functional heterogeneity of transferrin-bound iron: iron uptake by cell suspensions from bone marrow and liver and by cell cultures of fibroblasts and lymphoblasts.

According to the hypothesis of Fletcher and Huehns, functional differences exist between both iron-binding sites of transferrin. The site designated A should mainly be involved in the delivery of iron to erythroid cells, whereas site B should donate its iron preferentially to cells involved in the absorption and storage of iron. In the present study this hypothesis could be confirmed by in vitro experiments with various cell types. Iron transferrin preincubated with rat bone marrow cells donates less iron to rat bone marrow cells, Chinese hamster fibroblasts, human fibroblasts and human lymphoblasts than freshly prepared iron transferrin equal in iron and transferrin concentraion. Rat liver parenchymal cells, however, take up more iron from preincubated than from freshly prepared iron transferrin. Obviously, site A not only donates iron preferentially to erythroid cells but also to (rapidly) dividing nonerythroid cells in culture. From experiments with iron transferrin mixtures in which radioiron was present at low or high iron saturation, it could be concluded that rat bone marrow cells take up iron equally well from monoferric as from diferric transferrin. The observed functional heterogeneity could, therefore, not be ascribed to differences between monoferric and diferric transferrin.

Role of Transferrin in Determining Internal Iron Distribution

The behavior in vivo of transferrin in loading and unloading iron from its two sites was examined in rats. Radioiron entering the plasma from the gastrointestinal tract in iron-deficient, normal and iron-loaded rats did not differ in its subsequent tissue distribution between erythroid marrow and liver of normal recipients from a second isotope added to the same plasma in vitro. Loading studies in vitro were then carried out employing a reticulocyte incubation model designed to place one isotope predominantly on one site of transferrin, more available to the erythron, and the second isotope on the other site, more available to the liver. In 15 groups of animals in which 3 different iron salts were employed to load transferrin with iron, the mean isotope ratio in the erythron was 1.03 (+/-0.06 SD) and the mean liver ratio was 0.75 (+/-0.21 SD). It was found that the incubation of plasma with reticulocytes resulted in contamination of the plasma by radioactive hemoglobin. After allowance was made for hepatic uptake of radiohemoglobin in the 13 groups in which proper correction could be made, the isotope ratio in the liver became 0.97 (+/-0.17 SD). It is concluded that iron atoms from the two sites of transferrin have similar tissue distributions in vivo in the experimental situations examined.

Role of transferrin in determining internal iron distribution

The behavior in vivo of transferrin in loading and unloading iron from its two sites was examined in rats. Radioiron entering the plasma from the gastrointestinal tract in iron-deficient, normal and iron-loaded rats did not differ in its subsequent tissue distribution between erythroid marrow and liver of normal recipients from a second isotope added to the same plasma in vitro. Loading studies in vitro were then carried out employing a reticulocyte incubation model designed to place one isotope predominantly on one site of transferrin, more available to the erythron, and the second isotope on the other site, more available to the liver. In 15 groups of animals in which 3 different iron salts were employed to load transferrin with iron, the mean isotope ratio in the erythron was 1.03 (+/-0.06 SD) and the mean liver ratio was 0.75 (+/-0.21 SD). It was found that the incubation of plasma with reticulocytes resulted in contamination of the plasma by radioactive hemoglobin. After allowance was made for hepatic uptake of radiohemoglobin in the 13 groups in which proper correction could be made, the isotope ratio in the liver became 0.97 (+/-0.17 SD). It is concluded that iron atoms from the two sites of transferrin have similar tissue distributions in vivo in the experimental situations examined.

Iron dynamics in the ruminant

We have compared the plasma clearance rate of radioactive iron in cows both as ferric chloride and as iron specifically bound to transferrin. We have also repeated the transfusion experiment of Dern et al. (Dern, R.J., Monti, A. and Glynn, M.F. (1963) J. Lab. Clin. Med. 61, 280–291) using goats. The results show that neither non-specifically bound iron (Bates, G.W. and Schlabach, M.R. (1973) J. Biol. Chem. 248, 3228–3232) nor the iron bound to the two different sites in transferrin (Awai, M., Chipman, B and Brown, E.B. (1975) J. Lab. Clin. Med. 85, 769–784) can be identified as distinguishable iron pools by this technique.

Electron Paramagnetic Resonance Evidence for a Distinction between the Two Iron-binding Sites in Transferrin and in Conalbumin

Abstract The electron paramagnetic resonance spectra of aqueous solutions of both transferrin and conalbumin have been studied as a function of increasing sodium perchlorate concentration. Characteristic changes are observed which provide direct evidence for a distinction between the two metal-binding sites under certain conditions in the naturally occurring iron derivatives of each protein. This may be of physiological importance in the case of transferrin. Differences in the behavior of the two proteins were also observed.

Chemical modifications of the tryptophan groups of transferrin.

The tryptophan contents of human serum transferrin and human lactotransferrin have been estimated by colorimetric and spectrophotometric procedures. The nine tryptophan residues of human serum transferrin have been modified by 2‐nitrophenylsulphenyl chloride in 30% acetic acid, 2‐hydroxy‐5‐nitrobenzyl bromide at pH 3 and dimethyl‐(2‐hydroxy‐5‐nitrobenzyl)‐sulphonium bromide at pH 7.6. Upon modification under acidic conditions the iron‐binding capacity falls off linearly with degree of modification. At pH 7.6 no fall off in iron‐binding capacity was observed with a maximum of 8.4 2‐hydroxy‐5‐nitrobenzyl residues bound per molecule of transferrin. The results have been interpreted as showing that tryptophan residues are not directly involved in the iron‐binding sites of transferrin.

Study of the nature of the metal-binding sites and estimate of the distance between the metal-binding sites in transferrin using trivalent lanthanide ions as fluorescent probes.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTStudy of the nature of the metal-binding sites and estimate of the distance between the metal-binding sites in transferrin using trivalent lanthanide ions as fluorescent probesChun Ka LukCite this: Biochemistry 1971, 10, 15, 2838–2843Publication Date (Print):July 20, 1971Publication History Published online1 May 2002Published inissue 20 July 1971https://doi.org/10.1021/bi00791a006RIGHTS & PERMISSIONSArticle Views422Altmetric-Citations209LEARN 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 (620 KB) Get e-Alerts Get e-Alerts

Fluorescence and Absorption Studies of the Binding of Copper and Iron to Transferrin

A study has been made of the fluorescence and absorption changes that occur when Cu++ and Fe+++ bind to transferrin. Calculations of the overlap integrals and critical transfer distances involved show that the large fluorescence quenching produced by this binding is consistent with Forster long range energy transfer from tryptophan to absorption bands produced by the metal-protein interaction. A nonlinear dependence of fluorescence on metal binding was observed for iron but not for copper. This is also consistent with the Forster mechanism. Agreement between the experimental fluorescence results for iron binding and a phenomenological treatment with a random binding model is presented as evidence for independent and equivalent metal-binding sites in transferrin. Difference absorption spectra were obtained for the 250 to 600 mµ spectral range. Characteristic peaks in the region of tyrosine and tryptophan absorption indicated perturbation of these chromophores by the binding of metal ions.

The specific binding of iron(III) and copper(II) to transferrin and conalbumin

The binding of Fe 3+ to transferrin has been studied by pH titrations and equilibrium dialysis. In the pH range studied (7.5–8.9), 3 H + are released for each Fe 3+ bound. In the dialysis experiments, EDTA and citrate were used as competing chelating agents. It was found that the binding of Fe 3+ to transferrin may be described as coordination to two equivalent and independent sites. The nature of the binding sites in transferrin as well as conalbumin has been studied by measurements of ESR and visible spectra. The ESR spectra of the Fe complexes indicate that the metal is present as Fe 3+. Measurements of ESR and optical absorption as a function of the degree of binding in transferrin, coupled with the known binding constants, exclude strong interactions between the 2 Fe 3+, and indicate that the distance between them is greater than 9 Å. Attempts have been made to fit the experimental data to a spin-Hamiltonian and the results discussed in terms of the geometrical configuration of the complex. In the case of both Fe 3+ and Cu 2+, the ESR spectra of transferrin and conalbumin are very similar. The Cu 2+ spectra show hyperfine structure from 2–3 N nuclei. Urea denaturation destroys the specific bonding of Fe 3+ in transferrin, as shown by the parallel loss of color and ESR signal. The nature of the chelating sites in transferrin and conalbumin is discussed in relation to the present and earlier data. It is suggested that each chelating site involves 2 imidazole groups of the protein in addition to 3 tyrosyl residues.