The relative affinity of transferrin and albumin for zinc.

The relative affinity of transferrin and albumin for zinc has been measured by competitive dialysis at a low zinc concentration in 0.15 M NaCl, 50 mM HEPES, 0.1 mM trisodium citrate (pH 7.2). There were small differences between albumins and larger ones between transferrin preparations, but all albumins bound zinc more firmly than any transferrin did. It is known that transferrin is largely responsible for the uptake of zinc from an intestinal membrane in rats, but much of the metal is subsequently transferred to albumin. The current results show that both in humans and in rats (a) no special mechanism is needed to provide energy for this transfer, and (b) full equilibration would lead to virtually complete transfer in contrast with what actually occurs in vivo.

Competitive binding of iron by transferrins from different vertebrates.

1. A competitive dialysis technique has been used to study the relative affinities of the two iron-binding sites on transferrin molecules and the relative binding strengths of transferrins isolated from plasma of different species. 2. The comparisons were extended to include desialylated human transferrin, ovotransferrin, and a cyanogen bromide fragment of the latter. 3. Although the results of bilateral experiments could generally be accounted for in terms of the theory of independent sites, there were some exceptions, and cyclic comparisons were inconsistent. 4. All the comparisons made were compatible with a model in which site-interaction occurred, but it was not possible to decide whether the sites were intrinsically identical or not. For most species this corresponded to positive cooperativity, but for rabbit it was negative. 5. The average affinity of transferrin for iron depended on species, but the variation was never more than about one order of magnitude. 6. No effect on the binding constants for human transferrin could be detected when the sialic acid residues were removed. 7. The fragment of ovotransferrin competed fairly effectively with the native molecule for iron, although the average relative affinity was only about 1:15. 8. The relative binding of iron by ovotranferrin and human transferrin was affected little when bicarbonate anion was replaced by oxalate, although the ratio of the two binding constants for ovotranferrin increased.

Hepatic uptake and degradation of trace doses of asialofetuin and asialoorosomucoid in the intact rat.

Asialoorosomucoid and asialofetuin were prepared by using sialidase, which was removed chromatographically before the proteins were labelled with radioactive iodine. After intravenous administration of a small amount oa asialoglycoprotein (3--4 microgram/100 g body wt.) protein-bound and non-protein radioactivities in plasmas and livers of rats were determined at intervals over a period of 30 min. Transfer of either tracer protein from plasma to liver was almost complete in 5 min. Proteolysis of asialofetuin was evident very shortly thereafter, but degradation of asialoorosomucoid commenced after a significant delay and was initially slow relative to that of asialofetuin. Studies in vitro with crude hepatic lysosomal enzyme preparations indicated that asialoorosomucoid was less readily digested than asialofetuin, and that desialylation of orosomucoid or fetuin did not noticeably increase the susceptibility of these proteins to protease action. Proteolysis of asialofetuin was also demonstrable in liver homogenates in conditions under which albumin and asialotransferrin were stable. A generalized mathematical model was devised to represent the uptake and degradation of asialoglycoproteins by the liver. The theoretical assumptions that gave the best fits with experiment are outlined and discussed.

The physicochemical and chemical properties of alpha 1-acid glycoproteins from mammalian and avian plasmas.

We have isolated from the plasma of man, rabbit, pig, rat and chicken alpha 1-acid glycoproteins that show a single band on polyacrylamide gel electrophoresis and a single arc in immunodiffusion. Amino acid and carbohydrate compositions are presented. The human protein is very similar to preparations described by earlier workers and, although significant differences in amino acid composition exist among them, the proteins from the other species are assumed to be analogous to it. Ultra centrifugal studies, despite showing single, fairly symmetrical peaks at high speed, produced evidence for the physical heterogeneity of each protein in agreement with some earlier reports. Although many models consisting of mixtures of components were tested, none was found that fitted adequately all observations on any one of the glycoproteins.

Isolation of human beta1A-globulin by modern techniques.

1. Human beta(1A)-globulin was isolated from serum by precipitation with ammonium sulphate, gel filtration and electrophoresis in polyacrylamide gel. 2. The product was found by ultracentrifugation, analytical electrophoresis in polyacrylamide gel and two-dimensional immunoelectrophoresis to be of satisfactory quality for further study. 3. The amino acid composition of beta(1A)-globulin was determined. 4. In ordinary dilute buffers near neutrality, beta(1A)-globulin had S(0) (20,w) 6.42s and M 131 000, but some reversible aggregation occurred at lower pH. In neutral 6m-guanidine hydrochloride the molecular weight was not measurably different from that in dilute buffer.

The effect of rotor velocity on sedimentation coefficients.

Sedimentation-coefficient measurements on human IgG, fibrinogen and alpha(2)-macroglobulin and pig thyroglobulin were made at rotor velocities of 15220-59780 rev./min and 25.0 degrees C in capillary-type synthetic-boundary cells or ordinary cells. At the lowest velocity, IgG and fibrinogen gave results several per cent higher than at other velocities, whereas alpha(2)-macroglobulin and thyroglobulin gave values only about 1.5% higher. This behaviour of IgG and fibrinogen is attributed primarily to imperfect initial boundaries.

Differential sedimentation-velocity and gel-filtration measurements on human apotransferrin and iron-transferrin.

Differential measurements of sedimentation velocity showed that binding of 2 atoms of iron per molecule of human apotransferrin caused an increase in s(0) (20,w) of about 1.8%. Gel-filtration experiments to compare the elution volumes of apotransferrin and transferrin radioactively labelled with iron showed that binding of the first atom to a molecule produced a decrease in Stokes radius of about 0.7%, and the binding of a second atom an equal decrement. These results confirmed that saturation of human transferrin with iron alters the conformation sufficiently to produce detectable changes in the hydrodynamic properties. They also indicate that the local changes brought about by successive addition of 2 atoms of iron are very similar, if not identical.

Comparison of the sedimentation and gel-filtration behaviour of human apotransferrin and its copper and iron complexes.

Equilibrium-dialysis experiments showed that Tris or citrate in the solution prevented copper from occupying completely the specific metal-binding sites on human transferrin. Differential measurements of sedimentation velocity under conditions where two atoms of copper per molecule of protein were bound showed an increase in s(0) (20,w), relative to that of the apoprotein, practically the same as that produced by two atoms of iron. Gel-filtration experiments made under the same conditions to investigate the effect of copper binding on the Stokes radius of the protein showed merely that it lost most of the metal as it passed down the column.

Conformation changes and dissociation of Fc fragments of rabbit immunoglobulin G as a function of pH.

1. The sedimentation coefficients of rabbit immunoglobulin G, four types of Fc fragments, univalent Fab and bivalent F(ab)(2) fragments were measured as a function of pH. 2. In conjunction with molecular-weight determinations by sedimentation equilibrium, and with the behaviour on gel filtration, this enabled the state of association of the Fc fragments to be followed. 3. The type possessing an interchain disulphide bond, 1Fc fragment, changed extensively in structure, but not in molecular weight. 4. There was good correlation between the readiness to dissociate and the chain length of the shorter Fc fragments that do not contain the interchain covalent bond. 5. The increasing resistance to dissociation as the fragments became shorter ran parallel with the ability to resist enzymic attack. 6. The site of the strong association between component chains of Fc fragment is located in the C-terminal half. 7. The gel-filtration behaviour of the Fc fragments clearly confirms that the process is governed by the Stokes radius rather than molecular weight. 8. The ultracentrifugal results were used to estimate the separations of the hydrodynamic subunits in intact immunoglobulin G, and as a basis for a schematic structure.

The effects of cytotropic compounds on the resialylation of human asialotransferrin type 3 in the rat.

Effects of chloroquine, colchicine, leupeptin, taxol and vinblastine on the resialylation and degradation of human [125I]asialotransferrin type 3 were studied in rats. An improved experimental technique was applied that permitted the quantification of resialylated ligand produced by individual animals over 3 h by using deconvolution. All three microtubule inhibitors increased the proportion of the dose undergoing resialylation by 35-39%. In addition, colchicine, and, especially, vinblastine enhanced the overall recovery of the dose as protein-bound 125I. The dose recovery was also augmented by leupeptin without any concomitant change in resialylation. Chloroquine suppressed resialylation and this effect could only be partially lifted by the administration of colchicine. The blood of colchicine-treated rats possessed no resialylating activity toward the ligand even when supplemented with additional alkaloid in vitro. The observations support the view that the respective fractions of the ligand destined for resialylation and degradation can, to a certain extent, be varied independently of each other. The effects of short-term starvation (20 h) and refeeding (4 h) on these processes are also presented.

The subunits of thyroglobulin.

1. Pig thyroglobulin was reduced with dithiothreitol in the presence of sodium dodecyl sulphate, 8m-urea or 6m-guanidinium chloride. 2. The molecular weights of the reduction products were about 165000. 3. Two stable subunits with molecular weights as low as 35000 and 20000 were separated by Sephadex chromatography after prolonged exposure of the reduction products to sodium dodecyl sulphate at pH8.7. Although a hydrolytic reaction is probably implicated, the nature of the chemical linkages broken was not established.

Absorption of plasma proteins from peritoneal cavity of normal rats.

The present study was undertaken to examine whether the uptake of plasma proteins from the peritoneal cavity is quantitative so that tracers could be introduced that way for measuring their turnover. To this end, the metabolic behavior of seven homologous plasma proteins, labeled with 125I, was compared in rats after intravenous or intraperitoneal administration. The animals were maintained under physiological conditions. Total body radiation measurements showed that the degradation rates of albumin, immunoglobulins A and G, alpha 1-macroglobulin, and transferrin were the same regardless of the route of injection. This implies that these proteins are quantitatively absorbed from the peritoneum without undergoing modifications. The half-life of intraperitoneally injected alpha 1-acid glycoprotein was consistently shorter by an average 9%, thus suggesting that this protein becomes slightly altered if introduced that way. Only one-half of intraperitoneally injected fibrinogen survived normally, whereas the other underwent rapid degradation. The surviving molecules had the same half-life as fibrinogen injected intravenously. The fraction of surviving fibrinogen could be augmented by mixing the dose with serum. Within a wide range of concentrations and quantities injected, the degradation rate of transferrin remained the same. Analysis by deconvolution of the plasma curves of albumin and alpha 1-macroglobulin absorbed from the peritoneum showed that the transport process was independent of protein size and, at least up to 35 mg, of the amount injected. According to the same technique, intraperitoneally administered diferric transferrin retained its iron during passage into the circulation.

Partial resialylation of human asialotransferrin type 3 in the rat.

After the injection of a small dose (1 micrograms/100 g of body weight) of 125I-labeled human asialotransferrin type 3 in rats, the radioactivity became rapidly associated with the liver. However, during the ensuing 12 hr a significant fraction of the dose returned to the circulation as protein-bound 125I. The protein released by the liver was indistinguishable by gel filtration from the original preparation and was precipitable by an antiserum to human transferrin. Nevertheless, it no longer bound to the immobilized Gal/GalN-specific lectin from rabbit liver. However, binding could be restored to a large extent by treatment with neuraminidase, indicating that the loss of binding was due to resialylation. Changes in the electrophoretic mobility of asialotransferrin released by the liver showed that resialylation was partial--i.e., it involved the attachment of two or three sialyl residues. From analysis by deconvolution of the plasma curve of partially resialylated asialotransferrin it was calculated that the liver "repaired" this way approximately one asialotransferrin molecule out of four. Plasma clearance of partially resialylated asialotransferrin was similar to that of nondesialylated transferrin.