Interaction of human diferric transferrin with reticulocytes.

Abstract

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...

Basis of Plasma Iron Exchange in the Rabbit

“…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.…”

“…Transferrin was isolated from 275 ml normal rabbit plasma after iron saturation with ferrous ammonium sulfate under spectrophotometric control (3). 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).…”

“…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.…”

“…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.…”

Effects of Iron and Desferrioxamine on the Growth of Plasmodium Falciparum in Vitro

“…The labelling of the transferrin was always done in the presence of serum. FeS04 (0.1 M) is well known (Huebers et al, 1981) to be a very efficient donor of iron to transferrin. We also confirmed that, in our hands.…”