Transferrin continued

Fig. 3.161. (A) Zone electrophoresis patterns of FITC-labelled transferrin samples by fluorescence detection. The unbound dye (providing a main peak and several minor ones) was not removed from the samples. Experimental conditions background electrolyte, 100 mM borate buffer, pH 8.3 voltage, 20 kV capillary 59 cm (effective length 41 cm) X 75 pm i.d. injection of samples 100 mbar x s 20°C detection with fluorescence detector (240 - 400 nm, broadband excitation filter and a 495 nm cut-off emmision filter). The reaction was left to continue for 20 h, and the reaction mixtures contained 13 pm (1 mg/ml) Tf and (a) 0.01 mM FITC, (b) 0.1 mM FITC, and 1 mM FITC. (B) Zone electrophoresis patterns of an FITC-labelled transferrin sample by simultaneous fluorescence (upper trace, left axis) and UV detection (lower trace, right axis). The unbound dye shows several peaks with both detections. Experimental conditions background electrolyte, 100 mM borate buffer, pH 8.3 voltage, 20 kV capillary 59 cm (effective length fluorescence 41 cm, UV 50.5 cm) X 75 pm i.d. injection of samples 100 mbar X s 20°C detection with fluorescence detector (240 - 400 nm, broadband excitation filter and a 495 nm cut off emmision filter). The reaction was left to continue for 20 h, and the reaction mixtures contained 6.5 pm (0.5 mg/ml) Tf and 0.1 mM FITC. Reprinted with permission from T. Konecsni et al. [199].

A biological example of E° is the reduction of Fe(III) in the protein transferrin, which was introduced in Figure 7-4. This protein has two Fe(III)-binding sites, one in each half of the molecule designated C and N for the carboxyl and amino terminals of the peptide chain. Transferrin carries Fe(III) through the blood to cells that require iron. Membranes of these cells have a receptor that binds Fe(III)-transferrin and takes it into a compartment called an endosome into which H is pumped to lower the pH to —5.8. Iron is released from transferrin in the endosome and continues into the cell as Fe(II) attached to an intracellular metal-transport protein. The entire cycle of transferrin uptake, metal removal, and transferrin release back to the bloodstream takes 1-2 min. The time required for Fe(III) to dissociate from transferrin at pH 5.8 is —6 min, which is too long to account for release in the endosome. The reduction potential of Fe(IH)-transferrin at pH 5.8 is E° = —0.52 V, which is too low for physiologic reductants to reach. 

It should be pointed out, however, that not all hormones dissociate from their receptor in the pH 5.5 environment of the endosome [24], Some hormone-receptor complexes require much lower pH values for dissociation to occur. Although not a peptide hormone, the iron-transport protein transferrin is a peculiar example of this phenomenon and should be pointed out. In this case, at the neutral pH of the extracellular fluid transferrin containing bound iron binds to its cell surface receptor and is internalized. In the low pH environment of the endosome, iron becomes dissociated from transferrin, but transferrin remains bound to its receptor. The transferrin receptor, with bound transferrin, is then recycled to the cell surface. With iron no longer bound to the transferrin, the transferrin readily dissociates from its receptor at the neutral pH of the extracellular fluid [25,26]. This mechanism provides for an efficient continual uptake of iron into cells. Unlike transferrin, however, in those instances where peptide hormones have been documented not to be dissociated from their receptor in the endosome compartment, the hormone and receptor are delivered to the lysosomes via fusion of the endosomes with lyso-somes, where both hormone and receptor are degraded [24,27]. The continuous degradation of the receptor with each round of RME eventually leads to a decrease in the number of receptors on the cell surface, a phenomenon called down-regulation. 

Many studies have been conducted concerning variations in the transferrins in different animals. Buettner-Janusch (21), Barber and Sheeler (11, 12) and Kaminski (78) all participated in initiating studies with primates and work is continuing in this area from a variety of laboratories (56). Other studies on different species have included the following bovine transferrin (4, 5, 52, 57, 59, 134), sheep and goats (6), horses (19), and amphibia and reptiles (34, 47). 

The properties and biological importance of the transferrins can be expected to continue to generate expanding interest on the part of both physical and biological scientists. The many provocative facets of the comparative and genetic biochemistry should be obvious. Functionally, the serum transferrin is one of the few examples of a protein whose function is to chelate a metal ion in order to transport the metal ion rather than to form a catalytic complex with it. Not only must the transferrin form the complex and transfer the metal, but it must also release the... 

The binding assays that are performed in this flow injection system may be either competitive or sandwich type. An assay for the serum protein transferrin using a competitive ELISA (129) illustrates the principle (Fig. 5). An immunoafiinity purified polyclonal antiserum raised in rabbits against human transferrin is the binder. The immobilized antibody is packed in a small column (100-200 pL), which is placed in a continuous flow of buffer. The experimental setup is shown in Fig. 6. 

Oral iron therapy should not be given 24 h before i.m. injections begin and for 5 days after the last i.v. injection not only is continuation unnecessary, but it may promote adverse reactions by saturating the plasma protein (transferrin) binding capacity so that the injected iron gives a higher unbound plasma iron concentration than is safe. 

Deferoxamine is a highly selective chelator of iron that theoretically binds ferric (Fe +) iron in a 1 1 molar ratio (100 mg deferoxamine to 8.5 mg ferric iron) that is more stable than the binding of iron to transferrin. Deferoxamine removes excess iron from the circulation and some iron from transferrin by chelating ferric complexes in equilibrium with transferrin. The resulting iron-deferoxamine complex, ferrioxamine, is then excreted in the urine. Its action on intracellular iron is unclear, but it may have a protective intracellular effect or may chelate extramitochondrial iron. The parenteral administration of deferoxamine produces an orange-red-colored urine within 3 to 6 hours because of the presence of ferrioxamine in the urine. For mild to moderate cases of iron poisoning, where its use is unclear, the presence of discolored urine indicates the persistent presence of chelatable iron and the need to continue deferoxamine. The reliance on discolored urine as a therapeutic end point has been challenged because it is not sensitive and is difficult to detect. ... 

As noted earlier, transferrin saturation (i.e., serum iron level divided by the TIBC) is also useful in assessing IDA. Low values likely indicate IDA, although low serum transferrin saturation values may also be present in inflammatory disorders. Fortunately, the TIBC usually helps to differentiate the diagnosis in these patients a TIBC greater than 400 mcg/dL suggests IDA, whereas values below 200 mcg/dL usually represent inflammatory disease. With continued progression of IDA, anisocytosis occurs and poikilocytosis develops, as seen on peripheral smear and indicated by increased RDW. In rare cases, a bone marrow examination can be performed to assess bone marrow iron stores. Bone marrow examination reveals absent iron stores in IDA. Documenation of decreased hemosiderin can confirm the diagnosis of IDA. In microcytic anemias due to all other causes, iron stores are detectable. 

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