Copper, transferrin binding

Distinct differences are also seen when anions other than C032 are used. The crystal structure of oxalate-substituted diferric lactoferrin shows differences in the anion binding in the two sites in the C-site the oxalate is symmetric bidentate, whereas in the N-site it is asymmetric (193). When Cu2+ is the metal ion the oxalate binding differences become even more pronounced. Copper-transferrin binds oxalate only in its N-terminal site (91). Copper-lactoferrin and copper-ovotransfer-rin each bind two oxalate ions but binding occurs preferentially in the C-lobe (157,192). These different affinities mean that hybrid complexes can be prepared with oxalate in one site and carbonate in the other (92, 157, 192). The use of oxalate as synergistic anion gives rise to spectroscopically distinct sites for other metal ions also (171). 

Transferrins bind Fe2+ weakly and it is likely that a transferrin- Fe2+-HC03- complex formed initially undergoes oxidation to the Fe3+-C032- complex within cells and within the bloodstream. A conformational change closes the protein around the iron ions.56 In yeast the previously mentioned copper oxidoreductase encoded by the FET3 gene appears to not only oxidize Fe2+ but also transfer the resulting Fe3+ to transferrin. Ceruloplasmin may play a similar role in mammals.33... 

CD studies of metal complexes (iron, copper) with human serum transferrin showed bands in the visible region (114) [earlier work in this field, mainly by ORD measurements is cited in Ref. (114)]. The binding of cupric or zinc ions to apotransferrin had no significant effect on the far ultraviolet CD (ORD) spectra (114). Similar results were also reported for chicken ovotransferrin (115). However, there were differences in the aromatic and other spectral regions up to 1000 nm between the CD spectra of the two copper transferrins indicating dissimilarities in the respective metal-binding sites (115). 

Hypoproteinemia may result in low levels of serum calcium, ceruloplasmin, and transferrin. Because losses of iron are at most 0.5-1.0 mg/24 hr, even with the heaviest proteinuria, other factors must operate to produce iron deficiency and microcytic hypochromic anemia. Although the copper-binding protein ceruloplasmin is lost in the urine in nephrotic subjects and its plasma levels are low, plasma and red cell copper concentrations are usually normal. Zinc circulates mainly bound to albumin and also to transferrin, and thus the reported reduction zinc concentration in plasma, hair, and white cells in nephrotic patients is not surprising. 

The iron-binding protein of serum transferrin was found in fraction IV-3,4 of human plasma when the plasma was fractionated by low temperature ethanol fractionation procedures (31, 116). By further subfractionations, serum transferrin could be concentrated in Cohn fraction IV-7 (30, 125, 126). Cohn (30) first reported the properties of the isolated protein, which he called the 3i metal-binding protein since the protein had been found to bind copper, and possibly zinc, as well as iron. Holm-berg and Laurell (66) proposed that the protein be called transferrin on the basis that the principal function of the protein was associated with the transport of iron in serum and that it was not the major copperbinding protein in human serum. 

Human serum transferrin and chicken ovotransferrin have been reported to bind cobalt, iron, copper, zinc, and manganese. The iron complex is red with an absorption maximum at 465 mp.. Complexes of copper and manganese are yellow. Ulmer and Vallee (128) formed a complex with Mn3+ by standing for 12 hours while Inman (68) formed a complex by addition of hydrogen peroxide to a mixture of Mn2+ and the transferrins. Absorption spectra for three of the colored complexes of human serum transferrin are given in Fig. 5. Extinction coefficients are listed in Table 9. 

Although the binding of bicarbonate or carbonate in the iron complex of serum transferrin and chicken ovotransferrin has been established unquestionably, no such vigorous proof has been presented for the copper complexes. As will be discussed below, this is an important omission, because it is difficult to approximate the proposed formula for the iron and copper complexes without proof of the presence of bicarbonate or carbonate in the copper complex. In addition, there appear to be no investigations on the question of whether the carbonate or bicarbonate is required for the initial binding of iron, or for the formation of the colored complex. 

Like the studies with optical rotatory dispersion, studies with electron spin (paramagnetic) resonance not only have revealed important differences among the metal-free transferrins and their metal complexes but also have given insight into the nature of the binding sites and the structure of the complexes. Aasa et al. (1) reported on the iron and copper complexes of human serum transferrin and chicken ovotransferrin while Windie et al. 137) reported on human serum transferrin, human lacto-transferrin, chicken ovotransferrin, quail ovotransferrin, and turkey ovotransferrin. 

Fig. 20. A schematic diagram indicating the possible binding of copper and iron in either conalbumin or transferrin showing the similarity of the sites for the two metals and their symmetry as inferred from the EPR results. (Biochemistry 2, 1314 [1963]).

There are several modes of protection from the activity of available iron or copper in vivo. (Antioxidant action is discussed in more detail in Chapter 4). Apotransferrin binds iron(III) for transport and delivery to cells. It is its capacity as an iron-binding protein which renders it also able to function as an antioxidant by making iron(III) unavailable for participation in iron-catalysed radical reactions. Only about 30% of the iron-binding sites on the transferrin in human plasma are normally occupied in vivo (transferrin concentration 1.2-2.0mg/ml). The copper-containing protein caeruloplasmin (0.2-0.4 mg/ml) is... 

Whilst growth factors in the serum provide specific proliferative stimuli, studies with cultured cells have indicated other important components for cell proliferation [18]. For example, insulin is required to facilitate glucose and amino-acid uptake, and transferrin, which binds iron, makes it available to the cell. Serum is also believed to supply trace elements such as selenium, copper and zinc as well as fatty acids important for cell growth. Some serum components such as ascorbate, a-tocopherol, caeruloplasmin and albumin may serve important antioxidant functions [19]. 

In spite of its larger size, with an ionic radius of 0.80A (161), In3+ appears to bind to transferrin with an affinity close to that of Fe3+ (145). In3+ displaces Cu2+ from copper-saturated ovotransferrin, and the In3+ even remains bound in the presence of an added twofold excess of Fe3+. Indium-transferrin also migrates indistinguishably from iron-transferrin (145) and gives the same closed conformation, as judged by small-angle X-ray scattering (105). 

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