Sites Of Transferrin Research Articles

Rapid Screening and Estimation of Binding Constants for Interactions of Fe3+ with Two Metalloproteins, Apotransferrin and Transferrin, Using Affinity Mode of Capillary Electrophoresis

The interaction behavior of Fe3+ with transferrin and apotransferrin (iron-free form) was investigated in this study using affinity capillary electrophoresis. Change in the mass and charge of protein upon binding to the metal ion in the capillary tube led to variation in its migration time and was used to measure the noncovalent binding interactions by fast screening method. Acetanilide was used as the electroosmotic flow (EOF) marker to avoid possible errors due to the change in EOF during the experiment. The binding results were calculated from the mobility ratios of protein (Ri) and EOF marker (Rf) using the formula (Ri − Rf)/Rf or ∆R/Rf. For more comprehensive understanding, the kinetics of the interaction was studied and binding constants were calculated. Results showed that the Fe3+ displayed insignificant interaction with both proteins at lower metal ion concentrations (5–25 μmol/mL). However, transferrin exhibited significant interactions with the metal ion at 50 and 100 μmol/mL (ΔR/Rf = 0.0114 and 0.0201, resp.) concentrations and apotransferrin showed strong binding interactions (ΔR/Rf = −0.0254 and 0.0205, resp.) at relatively higher Fe3+ concentrations of 100 and 250 μmol/mL. The binding constants of 18.968 mmol−1 and −13.603 mmol−1 were recorded for Fe3+ interaction with transferrin and apotransferrin, respectively, showing significant interactions. Different binding patterns of Fe3+ with both proteins might be attributed to the fact that the iron-binding sites in transferrin have already been occupied, which was not the case in apotransferrin. The present study may be used as a reference for the investigation of protein-metal ion, drug-protein, drug-metal ion, and enzyme-metal ion interactions and may be helpful to provide preliminary insight into the new metal-based drug development.

Molecular mechanism investigation of the neutralization of cadmium toxicity by transferrin.

Cadmium adversely affects the biological function of the liver. Transferrin might be involved in the detoxification system of cadmium. However, owing to the lack of investigation of the molecular mechanism of cadmium conjugating to transferrin, the role of transferrin in cadmium detoxification in the liver and how transferrin undergoes conformational and functional changes upon cadmium binding are not clear. In this article, we demonstrated the potential role of transferrin in the protection of the mouse primary hepatocytes against cadmium toxicity. After the incubation of hepatocytes with 10 and 100 μM CdCl2, pretreatment with transferrin significantly attenuated the reduction of cell viability in comparison with the samples treated with CdCl2 alone. Furthermore, a detailed molecular mechanism investigation of the interaction of CdCl2 with transferrin was reported using biophysical methods. Multi-spectroscopic measurements showed that CdCl2 formed complexes with transferrin and caused structural and conformational changes of transferrin. Isothermal titration calorimetry measurements revealed that transferrin has two classes of binding sites with different binding constants for CdCl2 binding. Hydrophobic forces and electrostatic forces are the major driving forces of the interaction. Preferred specific binding sites on transferrin were identified by dialysis experiments, molecular docking studies and molecular dynamics simulations. Upon low CdCl2 concentration exposure, no content of iron was released from transferrin because CdCl2 preferentially binds to the surface of transferrin molecules. Upon higher CdCl2 concentration exposure, the release of iron content from transferrin was observed due to the interaction of CdCl2 with the key residues around iron binding sites.

N-Glycosylation Site Occupancy in Serum Glycoproteins Using Multiple Reaction Monitoring Liquid Chromatography-Mass Spectrometry

Congenital disorders of glycosylation (CDGs) are a family of N-linked glycosylation defects associated with severe clinical manifestations. In CDG type-I, deficiency of lipid-linked oligosaccharide assembly leads to the underoccupancy of N-glycosylation sites on glycoproteins. Although the level of residual glycosylation activity is known to correlate with the clinical phenotype linked to individual CDG mutations, it is not known whether the degree of N-glycosylation site occupancy by itself correlates with the severity of the disease. To quantify the extent of underglycosylation in healthy control and in CDG samples, we developed a quantitative method of N-glycosylation site occupancy based on multiple reaction monitoring LC-MS/MS. Using isotopically labeled standard peptides, we directly quantified the level of N-glycosylation site occupancy on selected serum proteins. In healthy control samples, we determined 98-100% occupancy for all N-glycosylation sites of transferrin and alpha(1)-antitrypsin. In CDG type-I samples, we observed a reduction in N-glycosylation site occupancy that correlated with the severity of the disease. In addition, we noticed a selective underglycosylation of N-glycosylation sites, indicating preferential glycosylation of acceptor sequons of a given glycoprotein. In transferrin, a preferred occupancy for the first N-glycosylation site was observed, and a decreasing preference for the first, third, and second N-glycosylation sites was observed in alpha(1)-antitrypsin. This multiple reaction monitoring LC-MS/MS method can be extended to multiple glycoproteins, thereby enabling a glycoproteomics survey of N-glycosylation site occupancies in biological samples.

Competitive Binding of Bismuth to Transferrin and Albumin in Aqueous Solution and in Blood Plasma

Several bismuth compounds are currently used as antiulcer drugs, but their mechanism of action is not well established. Proteins are thought to be target sites. In this work we establish that the competitive binding of Bi(3+) to the blood serum proteins albumin and transferrin, as isolated proteins and in blood plasma, can be monitored via observation of (1)H and (13)C NMR resonances of isotopically labeled [epsilon-(13)C]Met transferrin. We show that Met(132) in the I132M recombinant N-lobe transferrin mutant is a sensitive indicator of N-lobe metal binding. Bi(3+) binds to the specific Fe(3+) sites of transferrin and the observed shifts of Met resonances suggest that Bi(3+) induces similar conformational changes in the N-lobe of transferrin in aqueous solution and plasma. Bi(3+) binding to albumin is nonspecific and Cys(34) is not a major binding site, which is surprising because Bi(3+) has a high affinity for thiolate sulfur. This illustrates that the potential target sites for metals (in this case Bi(3+)) in proteins depend not only on their presence but also on their accessibility. Bi(3+) binds to transferrin in preference to albumin both in aqueous solution and in blood plasma.

Receptor mediated endocytosis and cytotoxicity of transferrin-mitomycin C conjugate in the HepG2 cell and primary cultured rat hepatocyte.

Intracellular disposition and cytotoxicity of macromolecular conjugate of mitomycin C (MMC) with transferrin (TF) were examined in the human hepatoma cell line HepG2 cell and normal cultured rat hepatocyte. The conjugate (TF-MMC) was specifically bound to the HepG2 cell as well as TF. The number of the binding site and the association constant of TF-MMC in the HepG2 cell were 396000+/-31000 molecules/cell and 3.24 x 10(7)+/-0.58 x 10(7) M(-1), respectively. No difference in the binding parameters of TF-MMC and TF can be detected in the HepG2 cell. The association constant for the TF receptor was almost identical between HepG2 cell and hepatocyte, however, the numbers of the binding site of TF-MMC and TF in the HepG2 cell were from 40-times to 50-times greater than those in the hepatocyte. Furthermore, TF-MMC was internalized into the HepG2 cell and the hepatocyte as well as TF. The rates of internalization of TF-MMC and TF into the HepG2 cell were nearly identical to those into the hepatocyte. However, the levels of the internalization into the HepG2 cell were remarkably higher than those into the hepatocyte because the number of receptors in the HepG2 cell was larger than that in the hepatocyte, and the rate of release from the HepG2 cell was slower than that from the hepatocyte. TF-MMC inhibited the growth of the HepG2 cells. The 50% growth inhibition (GI50) of TF-MMC against the HepG2 cell was 0.9 microg MMC/ml, which was a little higher than that of MMC (GI50=0.5 microg/ml). These results indicated that the TF-MMC might be useful for delivery of MMC to the HepG2 cell.

Transferrin Saturation and Screening of Genetic Hemochromatosis

Genetic hemochromatosis is the most frequent inherited disease in Caucasian populations. The gene located on the short arm of chromosome 6 was cloned in 1996 (1) and unfortunately named HLA-H. It encodes a protein that is very similar to HLA class I proteins. Two mutations have been described, but the missense mutation (C 282 Y) is observed in >90% of homozygous patients (2). The fact that the percentage of mutations is variable from one population to another implies the existence of other genes, and makes a genotypic screening impossible. In 1997 a consensus conference met in Atlanta to define the best procedure to be followed in screening for genetic iron overload. The assay of serum iron and total iron-binding capacity (TIBC) has been recommended as the first step of a phenotypic diagnosis. The ratio between serum iron and TIBC gives the percentage of transferrin saturation with iron. TIBC is an indirect measurement of the transferrin concentration. The first method described by Ramsay (3) was recently modified by the same author (4): Excess iron is added to serum to saturate both binding sites of transferrin, and non-bound iron is removed by adsorption on “light” magnesium carbonate. The adsorption capacity of MgCO3 is well known to vary from one preparation to another (4)(5). Checking the ability of each brand to adsorb iron is rather cumbersome. There is no reference method for TIBC; nevertheless, a selected method has been published (5). In 1990 the NCCLS proposed a method for the assay of serum iron and TIBC and will probably finalize it this year. In between-laboratory comparisons, different results were obtained, depending on the analytical procedure (6). In some pathological disorders, molecules such as ferritin or desferrioxamine bind added iron (7): TIBC …

Stereoselective interaction of drug enantiomers with human serum transferrin in capillary zone electrophoresis (II).

Further studies on chiral resolution of drugs with different chemical structures by capillary zone electrophoresis using iron-free human serum transferrin are described. The substances passed a highly concentrated pseudo-stationary protein zone applied in a coated capillary and the possible chiral separation of the optical isomers was followed. Eighteen drugs with different structures were screened, and the enantiomers of clofedanol, buphenine, acebutolol and chlorphenamine were resolved. Several, but not all drugs, showed longer migration times while passing the protein zone, indicating an interaction with transferrin, although chiral resolution was not observed in all cases. The observations provided further information about the properties of the surface interaction sites of transferrin.

Evidence for functional differences between the two sites of rabbit transferrin: Effects of serum and carbon dioxide

A recently developed technique combining urea gel electrophoresis with Western immunoblotting has been modified for assessing the relative ability of each iron binding site of rabbit transferrin in delivering iron to rabbit reticulocytes. The two sites can be made to release iron at the same or differing rates, depending on the experimental conditions. In Hanks' balanced salts solution in an atmosphere of room air or 5% CO 2, the acid-labile site in the N-terminal lobe of the protein was found to be 1.4- and 2.9-times more effective than its acid-stable counterpart in providing iron to reticulocytes after 90 min incubation. Both sequential and simultaneous release of iron from the two sites was observed, but sequential release was initiated only from the N-terminal site. The same site also proved to be a better iron donor by a factor of 2 when incubations were conducted in Hanks' medium enriched with 20% serum in 5% CO 2. Only in 20% serum in air were the two sites found to be equivalent iron suppliers to reticulocytes. In the cases studied, an atmosphere of 5% CO 2 increased 2-fold the effectiveness of iron donation by the acid-labile site to reticulocytes, while the presence of 20% serum enhanced the iron-donating ability of the acid-stable C-terminal site. Thus, the transferrinreticulocyte interaction is sensitive to environmental variables, and such sensitivity may help account for apparent discrepancies in previous studies of the relative iron-donating abilities of the two sites of transferrin.

Effects of anion binding on the conformations of the two domains of ovotransferrin.

A previous paper (Harris (1985) Biochemistry 24, 7412-7418) reported the occurrence of two classes of anion binding sites in transferrin. To evaluate the locations of the two anion binding sites in relation to the two major domains of transferrin we determined the binding constants of whole ovotransferrin and its two half-molecules by means of the difference UV spectroscopic technique. Anions induced strong negative absorbance at 245 nm in the order: citrate greater than phosphate greater than bicarbonate for whole ovotransferrin and the N-terminal half-molecule; and: phosphate greater than citrate greater than bicarbonate for the C-terminal half-molecule. The anion dissociation constants of the N-terminal half-molecule were consistent with lower dissociation constants, and those of the C-terminal half-molecule, with higher dissociation constants of whole ovotransferrin, indicating that the two classes of anion binding sites correspond to the binding sites in individual structural domains. Anion binding markedly protected the N-terminal half-molecule, but not the C-terminal half-molecule from digestion with trypsin and disulfide reduction with dithiothreitol. As to the far and near ultraviolet CD spectra data, however, there was no significant difference between in the presence and absence of an anion. Therefore, the binding of an anion would induce some conformational changes which were not reflected by the CD spectrum.

Specific binding sites for transferrin on ameloblasts of the enamel maturation zone in the rat incisor.

During enamel maturation in rodents, an iron-containing pigment is deposited into the surface layer of the enamel. Maturation zone ameloblasts presumably are responsible for this deposition. The presence of large amounts of ferritin in the cytoplasm of these cells suggests that they receive iron, presumably from circulating transferrin. An in vivo radioautographic binding assay using iodinated transferrin was used to determine if indeed maturation ameloblasts possess transferrin receptors at their cell surfaces. Experimental rats received systemic injections of labeled transferrin while control rats received injections of labeled transferrin plus a large excess of unlabeled transferrin in order to compete with the labeled transferrin for available specific receptors. Light microscope radioautography showed that ruffle-ended ameloblasts (RAs) of the enamel maturation zone had a high density of specific receptors for transferrin relative to smooth-ended ameloblasts (SAs). Electron microscopy and energy-dispersive X-ray spectroscopy confirmed the presence of ferritin and iron, respectively, within these cells. It is postulated that the iron responsible for enamel pigmentation is transported by transferrin to maturation ameloblasts and is bound to specific transferrin receptors found mostly on RAs and that the modulation of these cells into SAs results in a loss of most of these receptors.

Transferrin variants in sheep: separation and characterization by polyacrylamide gel electrophoresis and isoelectric focusing.

Isoelectric focusing with carrier ampholytes in ultrathin polyacrylamide gels and polyacrylamide gel electrophoresis in a discontinuous buffer system were used for the separation of sheep transferrin variants. For identification of the different iron-binding sites of transferrin a stepwise urea gradient, different degrees of iron saturation and double one-dimensional electrophoresis were used. Isoelectric focusing results in an increased resolution of the Fe0-transferrin, Fe1-transferrin and Fe2-transferrin region. At the level of Fe0-transferrin and Fe1-transferrin the variants I, A, G, B, C, D, M, E, Q, P can be identified. The method is especially suitable for genetic studies. For screening purposes up to 108 samples can be separated within one run in an ultrathin gel.

Interaction of transferrin with iron-loaded rat peritoneal macrophages.

Rat peritoneal macrophages are capable, in vitro, of processing and releasing iron derived from phagocytosed, immunosensitized red cells. From 20% to 60% of the red cell iron can be returned to the culture medium in 24 h, with resident macrophages more active than inflammatory, peptone-induced macrophages. When apotransferrin is present in the culture medium, from 39% to 72% of iron released from macrophages is bound to the protein, with most of the remainder in a ferritin-like form. No distinct preference of released iron for either site of transferrin could be observed. The absence of apotransferrin depresses iron release only slightly, with much of the iron then released in a form readily available to the protein in vitro. Pronase treatment of macrophages, which abolishes their ability to bind transferrin, depresses iron release no more than 10-15%. It appears, therefore, that binding of apotransferrin to macrophages may not be essential for iron excretion by the cells.

Complete inhibition of transferrin recycling by monensin in K562 cells.

Monensin blocks human transferrin recycling in a dose-dependent and reversible manner in K562 cells, reaching 100% inhibition at a noncytocidal dose of 10(-5) M, whereas transferrin recycling is virtually unaffected by noncytocidal doses of chloroquine. The intracellular pathway of human transferrin in K562 cells, both in the presence and absence of 10(-5) M monensin, was localized by indirect immunofluorescence. Monensin blocks transferrin recycling by causing internalized ligand to accumulate in the perinuclear region of the cell. The effect of 10(-5) M monensin on human transferrin kinetics was quantitatively measured by radioimmunoassay and showed a positive correlation with immunofluorescent studies. Immunoelectron microscopic localization of human transferrin as it cycles through K562 cells reveals the appearance of perinuclear transferrin-positive multivesicular bodies within 3 min of internalization, with subsequent exocytic delivery of the ligand to the cell surface via transferrin-staining vesicles arising from these perinuclear structures within 5 min of internalization. Inhibition of ligand recycling with 10(-5) M monensin causes dilated transferrin-positive multivesicular bodies to accumulate within the cell with no evidence of recycling vesicles. A coordinated interaction between multivesicular bodies and the Golgi apparatus appears to be involved in the recycling of transferrin in K562 cells. Cell-surface-binding sites for transferrin were reduced by 50% with 10(-5) M monensin treatment; however, this effect was not attenuated by 80% protein synthesis inhibition with cycloheximide, supporting the idea that the transferrin receptor is also recycled through the Golgi.

Metal-binding properties of a single-sited transferrin fragment

A single-sited iron-binding fragment of transferrin, prepared by proteolytic cleavage with thermolysin, has been characterized. The fragment, bearing no carbohydrate, must be derived from the N-terminal half of the protein's two homologous domains. The strength of iron-binding at pH 6.7 and 7.4, and the EPR spectroscopic features of its iron and copper complexes, establish it as carying the ‘b’ site of transferrin.

The two-sited nature of transferrin: Spectroscopic, thermodynamic and physiologic studies

Abstract The transferrins comprise a class of two-sited metal-binding proteins widely distributed in cells and fluids of vertebrates. Each of the major transferrin varieties—serum transferrin, ovotransferrin (conalbumin) and lactoferrin (lactotransferrin)—consists of a single polypeptide chain with extensive internal homology indicating evolutionary origin from a single-sited precursor protein. In human transferrin and hen ovotransferrin, at least, evolutionary development has altered the two sites so that they are chemically and spectroscopically distinguishable, but whether the differences between the sites are physiologically important is not yet clear. The metal-binding activities of all transferrins are dependent on the concomitant binding of stucturally-suitable anions. In human serum transferrin, anion- and metal-binding activities alike vary between the two sites. For example, the sites may be distinguished by the thightness with which they bind ion, by their accesibility to different chemical forms of iron, by their vulnerability to acid, by the rates at which they exchange anions with the medium, by their capacity to accomodate different anions when Cu(II) is the bound metal ion, and by their spectroscopic features. When single-sited transferrin fragments are prepared by selective proteolytic digestion, the tightness of ion-binding by these fragments is virtually identical to that of corresponding sites in the holoprotein.Evidently, therefore, site-site interactions are not responsible for the observed differences between the sites in native transferrin. Because each of the sites of transferrin resides in its own independent domain, and empty-sited domains are more susceptible to denaturation than occupied domains, each of the four possible molecular species of transferrin (apoprotein, monoferric-A and monoferric-B proteins, and differic protein) can be separated and displayed by electrophoresis in a 6M urea-gel system introduced by Makey and Seal. This procedure has been exploited to study the physiological iron-exchanging properties of the sites, in vitro and in vivo . In studies conducted in our laboratory, the two sites of diferric transferrin differ considerably in their ability to donate iron to isolated hemoglobin-synthesizing reticulocytes in an artificial culture medium. In in vivo studies in the rabbit, however, the two iron atoms are removed from diferric transferrin in pairwise fashion, so that the sites now appear equivalent in their iron-donating properties. Furthermore, iron, from stores and sites where it is absorbed or recovered from senescent red blood cells, is returned to empty sites of circulating transferrin, predominantly one atom at a time. Since studies of iron distribution among the sites of circulating transferrin in normal human subjects indicate that the sites are unequally occupied, with the more weakly binding site of the N-terminal half of the molecule favored, it seemed that the sites might differ not in their iron-donating abilities, but in their iron-accepting properties. Accordingly, the distribution of absorbed radioactive iron between the two sites of circulating transferrin was studied in the rabbit. In four of five animals the B-site was strongly favored by the newly absorbed iron. This may account, then, for the unequal distribution of iron between the sites of circulating transferrin. If the chemical differences between the two sites of transferrin are mapped into differences in function, therefore, it is probably on the ‘supply side’ of the iron economy.

Molecular aspects of the binding of absorbed iron to transferrin

To study the molecular aspects of the binding of absorbed iron to plasma transferrin, 59Fe with high specific activity was administered via intragastric tube to iron-deficient rabbits. The distribution of the absorbed 59Fe among the molecular forms of iron-transferrin was analysed using urea-polyacrylamide gel electrophoresis. Absorbed iron was bound to circulating transferrin one atom at a time. In four out of five animals, absorbed iron was predominantly bound to the site in the N-terminal domain of the protein. Thus, the two sites of transferrin may differ in their ability to load absorbed iron.

Distance between metal-binding sites in transferrin: energy transfer from bound terbium(III) to iron(III) or manganese(III)

The distance between the two metal-binding sites of human serum transferrin has been studied by observing energy transfer between an excited terbium ion bound at one site and a ferric (or manganic) ion bound at the other site of the same transferrin molecule. From the observed reduction in terbium lifetime (relative to that of terbium transferrin), it is concluded that the intersite distance is 3.55 +/ 0.45 nm. This distance is reconciled with two conflicting earlier reports that the separation between sites is greater than 4.3 nm [Luk, C.K. (1971) Biochemistry 10,2838-2844] or is equal to 2.5 +/ 0.2 nm [Meares, C.F., & Ledbetter, J.E. (1977) Biochemistry 16, 5178-5180]. The difficulty of accurately measuring the quantum yield of protein-bound terbium provides the principal source of uncertainty in these measurements.

No functional difference of the two iron-binding sites of human transferrin in vitro.

1. According to the Fletcher-Huehns hypothesis there exists a functional difference between the two iron-binding sites of transferrin. 2. The aim of the study presented was to evaluate this hypothesis in a homogeneous system, with human bone marrow cells and pure human monoferric transferrins A and B. 3. For this reason normal human bone marrow cells were incubated with human monoferric transferrin. The monoferric transferrins A and B were obtained by selective labelling at different pH of apotransferrin followed by preparative isoelectric focusing in granulated gels. The uptake of iron by the cell suspensions from monoferric transferrins A and B was equal. 4. In a heterogeneous but more active system for the removal of iron from human transferrin in vitro the two human monoferric transferrins did not show any significant functional differences. 5. No support for the Fletcher-Huehns hypothesis could be obtained.

The behavior of transferrin iron in the rat

The behavior of rat transferrin has been investigated employing acrylamide gel electrophoresis and isoelectric focusing. In vitro trace labeling with iron chelates at 30 min was 93%-98% effective, whereas binding by simple ferric salts was reduced to 71%-76%. Complete and specific binding of 59FeSO4 by the iron binding sites of transferrin was demonstrated after in vitro or in vivo addition of ferrous ammonium sulfate in pH 2 saline up to the point of iron saturation. In vitro the radioriron transferrin complex in plasma was stable and its iron had a negligible exchange with other transferrin binding sites over several hours. The distribution of radioiron added in vitro or through absorption was shown to be random between the binding sites of slow and fast transferrin molecule. Iron distribution among body tissues was similar for mono- and diferric transferrin iron and was not affected by the site distribution of iron on the transferrin molecule. The only important aspect of transferrin iron binding was the more rapid tissue uptake of iron in the diferric form was compared to monoferric transferrin. Additional in vivo effects on internal iron exchange were produced by changes in the iron balance of the animal. In the iron loaded animal, monoferric transferrin injected into the plasma was rapidly loaded by iron from tissue and thereby converted to diferric transferrin. Injection of diferric transferrin in the iron deficient animal was associated with a rapid disappearance from circulation of the original complex and a subsequent appearance of monoferric transferrin as a result of iron returning from tissues. These observations support the concept that plasma iron behaves as a single pool except that diferric iron exchange occurs at a more rapid rate than dose monoferric iron exchange.

Evidence for the functional heterogeneity of the two sites of transferrin in vitro.

A recently developed crossed immunoelectrophoretic method for displaying and quantitating the four possible molecular species of transferrin has been utilized to assess the relative effectiveness of each site of rabbit and human diferric transferrin in providing iron to rabbit reticulocytes. The site which appears to reside in the N-terminal half of the rabbit protein was found to be at least 5 times more effective than its counterpart. However, both sites may serve as iron donors in monoferric as well as diferric rabbit transferrins. It is also possible that iron may be removed from rabbit transferrin in pairwise as well as sequential fashion. In human diferric transferrin, the site in the C-terminal domain functions as the better iron donor for rabbit reticulocytes.