Methods have been devised for preparing human transferrin with a different isotope ofiron selectively labeling each of the two iron binding sites and for determining the distribution of radioiron among transferrin molecules. When diferric human transferrin was exposed to human or animal reticulocytes, there was an equal contribution of radioiron from the acid-stable and acid-labile sites. In this delivery, both atoms ofiron were removed simultaneously from the diferric transferrin molecule, converting it to apotransferrin. At similar iron concentrations the amount of iron delivered by diferric transferrin was twice that delivered by monoferric transferrin.Plasma transferrin mediates internal iron exchange in mammalian species. The protein has two binding sites for iron. In most species these sites differ somewhat in their chemical behavior in vitro, and there has been considerable discussion as to possible differences in behavior in vivo (1). This report describes methods whereby distribution of iron between the sites of human transferrin may be determined and characterizes the behavior of human diferric transferrin in donating its iron to human, rabbit, and rat reticulocytes. MATERIALS AND METHODSMaterials. The radioiron isotopes were purchased from New England Nuclear (59Fe and 55Fe) as ferrous sulfate, specific activity 13-22 ,uCi/,ug ofiron, dissolved in 0.5 M HC1 (1 Ci = 3.7 x 10"0 becquerels). Radioiodine for protein iodination was a product of Amersham (12'I as sodium iodide, specific activity 13-17 ,uCi/,ug of iodine). Nitrilotriacetic acid disodium salt was provided by Sigma (purity about 99.5%). Ferrous ammonium sulfate and other chemical reagents used were of analytical grade. Hanks' balanced salt solution was obtained from GIBCO.Isolation of Purified Human Transferrin. 59Fe-Tagged diferric human transferrin was isolated from the plasma of a healthy human volunteer as described elsewhere (2) except that the plasma was brought exactly to the point of saturation under spectrophotometric control (3). The resulting 59Fe-tagged diferric preparation had an absorbance ratio A465/A280 of0.045 and was found to be free of hemopexin. In order to obtain 59Fe diferric preparation of utmost purity, the transferrin preparation was subjected to isoelectric focusing on a preparative scale (for details, see Isoelectric Focusing Procedure).59Fe-Tagged monoferric transferrin carrying the iron at the acid-stable site of the molecule was prepared by column chromatography on Sephadex G-50 (column 2.5 x 50 cm, equilibrated with 0.115 M sodium phosphate buffer, pH 7.2). At least 200 mg of the 59Fe diferric transferrin preparation dissolved in 20 ml ofa 0. 1 M Tris-HCl buffer (pH 8.3) was put on the gel and eluted at room temperature with 0.115 M phosphate buffer (pH 7.2) at a flow rate of4 ml/min. Fractions with 5-ml volume were collected. The 59Fe fractions coming at the void volume of the column were pooled and subjected to analysis by spectrophotometry (2) and isoelectric focusing (4). The A4,J/Aaso ratio of the...
Normal dogs were treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) at 10 gg/kg/day for 30 d, which caused an initial neutrophilia, followed by a prolonged period of chronic neutropenia. A control dog treated with recombinant canine G-CSF (rcG-CSF) showed persistent neutrophilia over 3 mo. Serum from dogs during neutropenia contained an antibody to rhG-CSF, which neutralized the stimulatory effects of both rhG-CSF and rcG-CSF on dog marrow neutrophilic progenitor cell growth and on NFS-60 cell proliferation. 4 mo after discontinuation of rhG-CSF, the dogs' neutrophil counts returned to the normal range. Rechallenge with the rhG-CSF re-induced severe neutropenia in 1 wk. Neutropenia was transferred by plasma infusion from a neutropenic dog to a previously normal dog. These data suggest that human rhG-CSF immunizes normal dogs and thereby induces neutralization of endogenous canine G-CSF and neutropenia. This model system should allow more precise definition of the in vivo role of G-CSF. (J. Clin. Invest. 1991. 87:704-710.) Key words: immunoglobulin * in vitro culture * in vivo treatment
Purified fractions of human apotransferrin, monoferric transferrins with iron on the acid-labile binding site and on the acid-stable binding site, and diferric transferrin have been prepared. The iron loading and unloading behavior of these preparations has been examined by isoelectric focusing. Iron release from the two monoferric transferrin preparations to human reticulocytes was of similar magnitude. In a mixture containing equal amounts of diferric and monoferric iron, approximately 4 times the amount of iron delivered by the monoferric species was delivered by the diferric species. Iron loading of transferrin in vitro showed a random distribution between monoferric and diferric transferrin. Among the monoferric transferrins, loading of the acid-labile binding sites was greater than that ofthe acid-stable binding sites. In vivo iron distribution in normal subjects, as evaluated by in vitro-added 59Fe, gave similar results. Absorption of a large dose of orally administered iron in iron-deficient subjects resulted in a somewhat greater amount of diferric transferrin at low saturation and a somewhat smaller amount of diferric transferrin at higher saturations than would have been anticipated by random loading. These data would indicate that in the human, iron loading of transferrin may be considered essentially random. Unloading from the two monoferric transferrin species is of similar magnitude but far less than that delivered by diferric transferrin.Internal iron exchange in man is mediated by the plasma protein, transferrin. Methods for isolating and quantitating human transferrin fractions and the behavior of the diferric form in donating iron to reticulocytes have been described (1). In this report, the iron loading and unloading behavior of the two monoferric fractions along with the diferric fraction is described. MATERIALS AND METHODSTransferrin Preparations. Isolation of human diferric transferrin and its conversion to apotransferrin was as described (2,3). In this study two independent procedures were employed for the isolation of the two monoferric transferrins and the diferric form.Method 400 V and 4°C (3). This resulted in three distinct, equally colored bands representing, in order of their appearance from the acid side of the gel, diferric, monoferric acid-labile, and monoferric acid-stable transferrins (Fig. 1A). The gel was cut into slices of 2-mm thickness. The slices belonging to the individual transferrin bands were pooled, and their protein was eluted by overnight shaking in a water bath after the addition of0.1 M Tris'HCl (pH 8.3) (3). Measurement of radioiron content has been described elsewhere (2).Method 2. As starting material, 143 ml of plasma (with a serum iron concentration of 80 ,g/dl and an unsaturated ironbinding capacity of 270 ,g/dl) from a normal volunteer was used. Labeling with trace amounts of radioiron was achieved by the addition of 50 ,uCi of 59FeSO4 in 2 ml of 0.01 M HC1 to the well-stirred plasma over a period of2 min. After incubation for 30 min at 37°C, th...
A B S T R A C T Fast and slow rat transferrins were isolated by isoelectric focusing and prepared in their di-and monoferric forms. A comparison of the rates of iron release between fast and slow diferric transferrins and between fast and slow monoferric transferrins when incubated with reticulocytes or injected in vivo showed no significant difference in the behavior of the two isotransferrin species. Reticulocyte uptake of diferric transferrin resulted in the removal of both iron atoms from the transferrin molecule. A twofold greater iron uptake was observed from diferric as compared with monoferric iron, provided reticulocyte receptors were saturated. It is concluded that the two species of transferrin and their individual sites function similarly in their release of iron to tissue receptors.
Radioiron-and radioiodine-labeled forms of human diferric and monoferric transferrin and apotransferrin, isolated by preparative isoelectric focusing, were used to define transferrin-iron uptake by human reticulocytes. In mixtures of human diferric and monoferric transferrin, the diferric molecule had a constant 7-fold advantage in delivering iron to reticulocytes, as compared with the 2-fold advantage when single solutions of mono-and diferric transferrins were compared. This was shown to be due to competitive interaction in iron delivery, probably at a common membrane-receptor binding site for transferrin. Apotransferrin did not interfere with the iron-donating process and its limited cellular uptake was inhibited in noncompetitive fashion by diferric transferrin.Transferrin mediates iron exchange between body tissues. There has been considerable discussion concerning the behavior of this protein in view of the chemical differences between its two iron binding sites (1). Our recent studies, however, have shown that iron loading of the binding sites is essentially a random process and the release ofiron on incubation with erythroid cells is an "all-or-none" phenomenon (2-5). In addition, it was shown that iron delivery from the two monoferric transferrins is identical when they are incubated in vitro with reticulocytes or bone marrow cells (4,6) or injected in vivo into rabbits (7). In all these experiments, diferric transferrin proved to be a better donor than the single-iron-loaded molecule (2-5, 8), in variation with an earlier report (9). While part of this advantage relates to the simultaneous release of both iron atoms, an additional preference for the diferric form has been suggested but not quantitated (10). In this report, the relationship ofiron supplied to human reticulocytes from human mono-and diferric transferrin is examined at physiological levels of plasma iron. In addition, the effect of apotransferrin on the iron delivery process was studied. MATERIALS AND METHODS Transferrin Preparations. Purification and radioiodinationof human diferric transferrin by using "1I and 131I were done as described (3,4). Conversion of the transferrin into apotransferrin was accomplished by using desferrioxamine (4).For the preparation of di[mFe]ferric transferrin, 90 ,uCi of 55Fe (10,Ci/,ugas FeSO 1 Ci = 37GBq; NewEngland Nuclear) was added to 200 mg of t'I-labeled apotransferrin (100 ,uCi of "'lI dissolved in 5 ml of 0.05 M Tris-HCl, pH 8.0/0.01 M NaHCO3) and this was followed by the addition ofsufficient iron as ferrous ammonium sulfate (39 ,g of iron/ml in 0.01 M HCl) to exactly saturate the free iron binding sites present in the transferrin solution. * This goal was achieved by spectrophotometric titration techniques in which the increase in color development at A = 465 nm was observed (11). At the point of saturation, the A465/Am ratio of the transferrin solution was 0.045, which is characteristic of the diferric state of the transferrin molecule (2, 3). Transferrin solutions were prepared at iron s...
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