Transferrin oxalate

Early experimental evidence in support of the hypothesis that an attack on the anion is at the heart of the iron-exchange mechanism (53) was soon corroborated by work from several laboratories (54, 55, 88). Replacing carbonate with oxalate at the specific anion-binding site of transferrin results in a relatively stable ternary Fe(III)-transferrin-oxalate complex. Over the time course of many hours or even days the oxalate complex slowly reverts to the physiologic Fe(III)-transferrin-carbonate form, but since in vitro studies seldom require more than an hour or two, the biologic properties of the oxalate complex can be tested. 

Figure 6. Uptake of iron by reticulocytes from 59Fe-transferrin-carbonate (O—O) and 59 Fe-transferrin-oxalate (X—X) (54)...

Fig. 26. Binding modes for anions other than carbonate. In (a) the mode of binding of oxalate to human lactoferrin, as determined crystallographically (192,193), is shown. In b is a generalized model for synergistic anion binding to transferrins, based on EPR studies (191) and the crystallographic results for oxalate. From Shongwe et al. (192), with permission.

Some tentative conclusions about the nature of these conformational differences may be drawn from the crystallographic studies of Cu2+ and oxalate-substituted lactoferrins (26,192,193). Anions which gave class A spectra with V02+-substituted transferrins are those that can... 

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). 

Intestinal absorption of is low, ranging from 0.4% to 2.5%, so fecal output is mainly unabsorbed dietary chromium. Absorption is increased marginally by ascorbic acid, amino adds, oxalate, and other dietary factors. After absorption, chromium binds to plasma transferrin with an affinity similar to that of iron. It then concentrates in human liver, spleen, other soft tissue, and bone. Urine chromium output is around 0.2 to 0.3 U,g/day, the amount excreted being to some extent dependent upon intake. Paradoxically, urine output appears to be relatively increased at low dietary levels. Thus 2% is lost in urine at an intake of lOpg/day, but only 0.5% at an intake of 40pg/day. Both running and resistive exercise increases urine chromium excretion. 

Figure 6. Energy levels and energy separations at selected molecular orientations. The energies calculated are part of simulations of transferrin oxalate EPR spectra. The molecular axes are oriented with respect to the magnetic field ai(0, = (49°,46.5°). Transition between levels 2 and 3 is highlighted by dotted lines. Levels are numbered as in Figure 3, and parameters of the calculatirm are given on the figure. The horizontal line in the lower panel is drawn at an X-band frequency of 9.23 GHz. The figure is reproduced fi-om Fig. 5 of Gaflhey and Silverstone [21], with permissirai of the publisher.

The relaxation times of enterobactin [27] can be compared with relaxation times of other biological non-heme, S = 5/2, iron examples, transferrin earbonate, and oxalate [28]. The observed EPR linewidths (>100 MHz) [13] of these nonheme iron proteins exhibit little temperature dependence (4-100 K), in contrast to observed widths of heme proteins. The measured phase memory times T T2) of the transferrins correspond to relaxation-determined linewidth contributions of <2 MHz in the range 4-30 K. The values of enterobactin bound to FepA indicate similarly small relaxation-determined widths. The apparent widths of these nonheme-iron EPR spectra therefore have an origin other than relaxation. 

Baker HM, Anderson BF, Brodie AM, Shongwe MS, Smith CA, Baker EN. 1996. Anion binding by transferrins importance of second-shell effects revealed by the crystal structure of oxalate-substituted diferric lactoferrin. Biochemistry 35(28) 9007-9013. 

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