Uptake and release of iron from human transferrin.

Abstract

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...

Basis of Plasma Iron Exchange in the Rabbit

“…Iron uptake must therefore occur one atom at a time; otherwise there would be a disproportionate amount of diferric transferrin present as compared with the proposed random distribution found. This same in vitro and in vivo loading pattern has been observed in other species (4,15). The release of iron from transferrin to reticulo- Figs.…”

“…The separation procedure by means of gel chromatography on Sephacryl S200, ion exchange chromatography on DEAE Sephacel, and conversion of the diferric into the apotransferrin form was the same as described for human transferrin (4). Isolation of apo-, mono-, and diferric transferrin was carried out by isoelectric focussing procedures identical to those described for human transferrin (3,4). In this instance, the starting material was 200 mg of rabbit apotransferrin one-third saturated with 55Fe or 59Fe-tagged ferrous ammonium sulfate.…”

“…The uptake of iron from transferrin by reticulocytes during in vitro incubation was studied (3,4). To produce reticulocytosis, a rabbit was bled 60 ml from the ear artery on days 1, 3, and 4 and blood obtained for the study on day 6.…”

“…To produce reticulocytosis, a rabbit was bled 60 ml from the ear artery on days 1, 3, and 4 and blood obtained for the study on day 6. The reticulocyte count of that blood was 29% and the hematocrit was 22.…”

“…Recently, methods have been developed (3,4) that make possible preparation of pure fractions of apo-, mono-, and diferric transferrin. Analytic procedures including both isoelectric focussing and gel electrophoresis permit quantitation of the different species of transferrin as they exist in the plasma.…”