Manduca sexta larvae accumulate large amounts of iron during their larval feeding period. When 59Fe was fed to 5th instar larvae, it was evenly distributed among the hemolymph, gut and carcass until the cessation of feeding. By pupation 95% of the labelled iron was found in the fat body. In the adult a significant portion of this iron was found in flight muscle. Studies of the hemolymph disclosed two iron-containing proteins. The first was composed of a single polypeptide chain of 80 kD, containing one atom of iron. This protein bound ionic iron in vitro and was able to transfer this iron to ferritin when incubated with fat body in vitro. Therefore, it appeared to serve a transport function. The second protein had a molecular weight of 490 kD with subunits of 24 and 26 kD and contained 220 micrograms of iron/mg protein. Its chemical and ultrastructural characteristics were those of ferritin. These studies demonstrate the presence of both a transport protein and a unique circulating ferritin in Manduca sexta, the latter serving a storage function during development and possibly also a transport function.

Two iron-binding proteins were isolated from rat intestinal mucosa. From determination of their molecular weights, their electrophoretic and iron-binding properties it was established that one was a mucosal ferritin and the other a mucosal transferrin.The mucosal ferritin is compared in its molecular weight, isoelectric point, amino acid composition and tryptic peptide pattern with the ferritins of rat spleen and liver. All three ferritins are distinctly different from one another. In addition the iron content of mucosal ferritin was found to be much lower than that of liver and spleen ferritins.Mucosal transferrin was separated into two components by isoelectric focusing, as was plasma transferrin. The plasma and mucosal transferrins differ in their isoelectric points and in their amino acid compositions. Differences were also found in vitro in the iron-binding of mucosal transferrin as compared with plasma transferrin. The role of these mucosal proteins in the absorption of iron is briefly discussed.

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...

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...