George W. Bates

Non-Specific Serum Iron in Thalassaemia: an Abnormal Serum Iron Fraction of Potential Toxicity

Summary. Iron binding in the sera of 35 patients with β thalassaemia major and intermedia was studied. In patients receiving regular blood transfusions since infancy transferrin was completely saturated and about 2.7‐7.I μmol/1 of the serum iron could be removed by dialysis or ultrafiltration in the presence of a chelating agent or by filtration on DEAE‐Sephadex‐catecholdisulphonic acid columns. In contrast, less than 1.0 μmol/1 of transferrin bound iron was removed when subjected to the same procedures. The non‐specific iron of thalassaemic sera could no longer be demonstrated after incubation with normal serum. These findings indicate that non‐specific iron is a chelatable compound which is readily available for transferrin binding. In view of the known toxicity of unbound iron, its identification in thalassaemic sera might be of relevance to the pathogenesis of tissue damage and the protective effect of iron chelating therapy in this disease.

Does transferrin exhibit ferroxidase activity

Abstract The oxidation of Fe2+ to Fe3+ by oxygen at pH 7.45 is a first order reaction with a 25 minute half life. In the presence of apotransferrin the oxidation rate is greatly enhanced and Fe3+-transferrin is formed. The apotransferrin mediated reaction reaches 50% completion in one minute; it does not follow simple first order kinetics. Iron-saturated transferrin does not exhibit the rate enhancement effect suggesting that the specific metal binding sites are the loci of the iron oxidation. Addition of H2O2, an agent which rapidly oxidizes Fe2+ to Fe3+, during the reaction of Fe2+ with apotransferrin greatly decreases the yield of Fe3+-transferrin. It is postulated that the basis of the rate enhancement effect is the binding of Fe2+ to the metal binding site of the transferrin molecule, followed by a rapid oxidation of the iron to the trivalent form.

Mobilization of iron from reticulocyte ghosts by cytoplasmic agents.

Summary The preparation of ghosts from rabbit reticulocytes previously incubated with 59 Fe-transferrin, allows the study of membrane iron mobilization by cytoplasmic factors, and intracellular iron pathways in a cell free system. Incubation of 59 Fe-ghosts with unlabeled reticulocyte lysate results in mobilization of the membrane bound iron, and its utilization for hemoglobin and ferritin synthesis. An iron binding component migrating near the low molecular weight range on a Sephadex G-100 column was rechromatographed on G-25 and emerged with the void volume. Chromatographic behavior suggested a molecular weight near 5,000. The component was subjected to gel electrophoresis and migrated as a single TCA precipitable band that stained with Coomassie blue. A Lowry test for protein was positive. The component was found to reversibly bind ferrous ion, and to be metabolically active.

The purification of lactoferrin from human whey by batch extraction

The isolation of lactoferrin from human whey has been accomplished using a rapid two-step procedure. The lactoferrin is directly adsorbed to cellulose phosphate by batch extraction and eluted by a stepped salt and pH gradient. The major impurity, a low-molecular-weight fraction, is quickly removed by gel filtration. The recovered lactoferrin has a purity of about 96%. The yield of lactoferrin averaged 80%. This method of lactoferrin purification greatly reduces the labor and time required, and the procedure is easily scaled to any volume of starting material.

The effect of serum and experimental variables on the transferrin and reticulocyte interaction.

The transfer of iron from transferrin to the developing erythrocyte is a research area of high interest and considerable controversy. We have found that the results of transferrin-reticulocyte incubation studies are quite sensitive to the experimental procedures that are utilized. Reticulocytosis has been induced in rabbits by phelbotomy and phenylhydrazine injections. While the latter gives a higher reticulocyte count, the cells appear to exhibit an altered transferrin-membrane interaction. Transferrin has been iodinated by published methods utilizing chloramine-T and molecular iodine. The iodotransferrin products exhibit the same iron donation ability, however, evidence was found that the chloramine-T treatment leads to a nonspecific binding of transferrin to the reticulocyte. The means of saturating transferrin with 59Fe is also of prime importance. Fe(NH4)2(SO4)2 and especially FeCl3 were found to yield nonspecifically bound iron when added to transferrin or serum. This artifact was reflected in an altered transferrin-reticulocyte interaction. Using what we believe to be optimal conditions, the effect of serum on the transferrin-reticulocyte system was re-examined. The results clearly indicated an enhancement of iron uptake by reticulocytes in the presence of serum, as well as an accelerated incorporation of iron by the cytoplasmic fraction.

A study of the anion binding site of transferrin.

Studies of the interaction between carbonate and iron binding by transferrin have centered about attempts to form a specific Fe3+-transferrin complex in the absence of carbonate and carbonate substitutes. Despite intensive experimental effort in our laboratory and others, such a complex has not been formed and verified [l--S]. A complicating factor is the propensity of Fe3+ to polymerize and bind in a non-specific fashion to the transferrin molecule [6], however, the continuing negative results clearly indicate that in the absence of suitable anions, transfer& has a low affinity for Fe 3+. Another unsolved question is the form of the anion, i.e. carbonate or bicarbonate. We favor carbonate on the basis of potentiometric data [5] and the assumption that the tight binding of the anion is achieved via strong electron withdrawal. This would lead to a decrease of the second acid pK through inductive effects and consequent release of the proton in question. The nature of the anion binding may be approached through a study of the stability, exchange rates, and physical parameters of Fe3+-transferrin-anion complexes [ 1,3]. We report here the preparation of an Fe3*-transferrin-14C03 complex, free of unbound radioactive carbonate. The exchange of carbonate at the anion binding site follows first order kinetics with a half life of one week. Nitrilotriacetate (NTA) may act as a carbonate substitute [7] and the stability of Fe’+--transferrin-NTA has been shown to be much lower than that of the physiological complex. The results are correlated with the transferrin literature and a new model for the binding of carbonate and iron to transferrin is proposed.

Failure of metallothionein to bind iron or act as an iron mobilizing agent

Abstract There are reasons to suggest metallothionein might act as a transient intracellular iron transport agent. Rabbit kidney metallothionein was isolated from animals treated with Cd in order to induce metallothionein synthesis. Attempts were made to form the iron-protein complex via several reaction routes. There was no evidence for complex formation. Metallothionein also failed to mobilize iron from ghosts of rabbit reticulocytes specifically labeled with 59 Fe.

Polyacrylamide gel staining with Fe2+-bathophenanthroline sulfonate☆

Abstract The utilization of Fe2+-bathophenanthroline sulfonate for the detection and quantitation of protein bands in cylindrical polyacrylamide gels is described. Two procedures are outlined. The first procedure is used in standard disc electrophoresis and involves fixing the protein with trichloroacetic acid, staining with Fe2+-bathophenanthroline sulfonate, and destaining with an ethanol:acetic acid solution. The second protocol reported is utilized with sodium dodecyl sulfate-containing gels. After electrophoresis, the gels are incubated with a methanol: acetic acid solution to remove the sodium dodecyl sulfate. The gels are then stained with Fe2+-bathophenanthroline sulfonate and destained with a methanol: acetic acid solution. Excellent background clarity is observed with both methods. Densitometric areas of the stained protein bands are linear to 60 μg of bovine serum albumin, and the limit of detection of this protein is 1 μg. Because of its rapidity of staining and destaining, good sensitivity, and reproducibility of stain intensity, Fe2+-bathophenanthroline sulfonate is an excellent protein stain.

Complex formation, polymerization, and autoreduction in the ferric fructose system

Abstract The solution chemistry of ferric fructose has been examined at various hydroxide-to-iron ratios using several physical techniques. The reactivity of ferric fructose complexes with transferrin, the serum iron binding protein, has provided a quantitative measure of polymeric fractions and insight into the rate and mode of breakdown of the polymers. A hydroxy-iron polymer is formed maximally (90%) in the neutral pH region corresponding to 3 OH/Fe, while mononuclear ferric fructose complexes predominate both in acidic (probably as FeFru(-H+)2+, and basic (probably as FeFru(-4H+)− solutions. The chemistry is similar to that of the ferric gluconate system, except that the carboxylate group of gluconate makes it more effective at breaking down the hydrolytic iron polymer. Both ligands form octahedral mononuclear complexes, and gluconate exhibits higher ligand field strength than fructose. The hydrolytic ferric fructose polymer shows the same spectrum as the ferric nitrate and ferric citrate polymers but is much smaller (mw

In vitro studies of iron bioavailability

The bioavailability of iron from foods is ultimately determined by interactions between iron and other components in the digestive milieu. Perhaps the most important factor is the concentration of Fe2+ during transit through the duodenum. During in vitro simulations of human digestion it is possible to probe the concentration of Fe2+, the rate of Fe2+ formation, and total iron concentration using ferrous chromogens. It is crucial, of course, that the chromogen not interfere with the redox reactions occurring during digestion. Accordingly, ferrozine was examined with regard to its ability to reduce complexes Fe3+, alter rates of Fe3+ production, detect Fe2+ present in the digestive mixture and differentiate the effects of chelating and reducing agents in the mobilization of iron from pinto beans. The chromogen was found to be free from apparent artefacts and to be a sensitive and reproducible probe of the state of iron in digestive mixtures.