Transferrins spectra

FIGURE 5.8 Complex hyperfine patterns due to axes noncolinearity in a low-symmetry prosthetic group. The X-band spectrum is from 65Cu(II)-bicarbonate in human serum transferrin (a,b) experimental spectrum (c,e) simulation assuming axial symmetry (d, f) simulation assuming triclinic symmetry with the A-axes rotated with respect to the g-axes over 15° about the gz-axis and then 60° about the new y -axis. Traces b, e, and f are 5x blow-ups of traces a, c, d, respectively (Hagen 2006). (Reproduced by permisson of The Royal Society of Chemistry.)... 

A cobalt complex of transferrin has been prepared by addition of Co(II) citrate to the apoprotein. Hydrogen peroxide was added to obtain the absorption spectrum of cobalt transferrin, and susceptibility measurements showed that the metal ion was incorporated as diamagnetic Co (III) (142). 

Fig. 17. Spectral shifts of iron complexes caused by succinylating ovotransferrin to varying degrees. The protein was succinylated in the iron-free state. Following dialysis and lyophilization, the modified transferrins were redissolved and saturated with iron, and the visible spectrum was scanned with a Beckman DB recording spectrophotometer. ------, original ovotransferrin (Xmax 465 mg) ---------, succiny-...

Follow the procedure in Subheading 3.2. However, in step 4 just prior to the addition of the ligand solution, add 100 ig/mL transferrin-AlexaFluor-647 or 300 (rg/mL dextran-Alexa Fluor 647 to the peptide/DNA mixture to visualize clathrin-dependent endocytosis or macropinocytosis, respectively. Analyze the cells either live or fixed and counter-stained. If the cells are fixed and counter-stained, omit adding TO-PRO-3 under step 18, as the fluorescence of TO-PRO-3 is in the far-red spectrum just as Alexa Fluor 647. 

The lanthanides modify the UV absorption spectra of proteins when aromatic chro-mophores are present at the binding site. Sharp maxima at 245 and 295 nm in the difference spectrum of transferrin saturated with Tb3+ ion as opposed to that of metal free protein were observed [20]. The shape of the spectrum is suggestive of lanthanide induced... 

The J sublevels of rare earths are split by ligand fields of low symmetry. These splittings may be observed in circularly polarized luminescence (CPL) but not in the fluorescence spectrum. Hence CPL spectra provide information on the rare earth ion and thus throw light on the binding site. The emission and CPL spectra of Tb3+ bound to transferrin and cobalbumin were found to be similar leading to the conclusion that the structure and... 

Fig. 20. The EPR spectrum of 65Cu2+-transferrin-carbonate. The inset shows a low-field hyperfine line (at an eightfold increase in gain) that displays superhyperfine splitting resulting from the coordination of one nitrogen ligand in each site. Adapted from Zweier and Aisen (91), with permission.

No C resonance was observable [93] from H COl bound to transferrin (mol. wt. 78000). Evidently, the Fe-C distance is less than about 9 A, and the resonance has broadened out. Transferrin tyrosyl resonances are also broadened by (high-spin) Fe in the H spectrum of the protein [94], which may indicate the protein-binding groups for iron. 

The spectrophotometric technique exploits the fact that when it binds to transferrin, AF+ replaces hydroxyl protons from two tyrosines, thus causing a change in the UV region of the spectrum (Fig. 10). Titration of the spectral change as a function of [AP+] at constant transferrin concentration allows the binding stoichiometry and stability constant to be measured. This approach has been used to study the interaction of a large number of metals [e.g., Nd +, Sm , Zn +, and Ga 61, 62,136)] with transferrin, including AP+ (33, 43, 136, 138) some of these metals would otherwise be spectroscopically silent. Such... 

Optical spectra of transferrin C-lobe docked with the transferrin receptor showed a characteristic broad absorption band centred at 465 nm, just as in the receptor-free /zo/o-protein (Figure 2.1 inset). The intensity of this absorbance band declined as more negative potentials were applied in a spectroelectrochemistry experiment, but did not qualitatively change in its overall features. An EPR spectrum of the Fec/TfR complex at pH 5.8, recovered from the OTTLE cell after completion of spectroelectrochemical studies allowed us to conclude that the first coordination shell of Fe " in transferrin is intact and unperturbed when C-lobe is complexed with TfR. Consequently, we assume that C-lobe and Fec/TfR complex have similar if not identical Fe " and Fe binding constants, and so we take for binding of Fe " in the protein-receptor complex to be 10 M as calculated for free Tf. This value was used to correct the observed Nernst plot data by accounting for the dissociation of Fe that occurs upon reduction. Nernst plots for the observed spectroelectrochemical data for FccTf/TfR, and data corrected for Fe dissociation, are presented in Figure 2.7. The corrected plot exhibits typical Nernstian behaviour for a one-electron transfer and a E1/2 value of —285 mV. 

Figure 19. S-band data for bound to transferrin, carnosine, hemoglobin and serum albumin. Top panel three-line spectrum for M, = -1/2 line in gn region indicates one donor nitrogen atom bound to cupric ion. Second panel expanded spectra (b) and second harmonic (c) for M,= —1/2 line in region indicates four equivalent nitrogen donor atoms. Third and bottom panel nine-line spectra attributed to four approximately equivalent nitrogen donor atoms (see text for further analysis). From [233,262,266], with permission.

Fig. 14. Mossbauer data for transferrin in variable conditions and the calculation from the electronic model. Each spectrum on the right was calculated with the assumption of a small randomly oriented field acting on the electronic moment. The data on the right were collected in exactly zero applied field...

A question of recent interest is whether the two iron sites in each molecule are equivalent (58), Mossbauer effect studies (28) and ESR studies (59) of human serum transferrin could detect no difference in the spectra of the two iron sites, but recently a very careful study by Aasa (27) has shown small differences in the ESR spectrum due to a small change in the ESR parameter A. The Mossbauer data were shown to be in good agreement with a calculation by Spartalian and Oosterhuis (28) and more recently by Tsang, Boyle and Morgan (60). The spin Hamiltonian parameters for each experimental study are presented in Table 4. 

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