The electrophoresis of transferrins in urea/polyacrylamide gels

The denaturation of transferrin by urea has been studied by (a) electrophoresis in polyacrylamide gels incorporating a urea gradient, (b) measurements of the loss in iron-binding capacity and (c) u.v. difference spectrometry. In human serum transferrin and hen ovotransferrin the N-terminal and C-terminal domains of the iron-free protein were found to denature at different urea concentrations

Studies of the binding of different iron donors to human serum transferrin and isolation of iron-binding fragments from the N- and C-Terminal regions of the protein

1. Trypsin digestion of human serum transferrin partially saturated with iron(III)- nitrilotriacetate at pH5.5 or pH 8.5 produces a carbohydrate-containing iron-binding fragment of mol.wt. 43000. 2. When iron(III) citrate, FeCI3, iron(II) ascorbate and (NH4)2SO4,FeSO4 are used as iron donors to saturate the protein partially, at pH 8.5, proteolytic digestion yields a fragment of mol.wt. 36000 that lacks carbohydrate. 3. The two fragments differ in their antigenic structures, amino acid compositions and peptide 'maps'. 4. The fragment with mol.wt. 36000 was assigned to the N-terminal region of the protein and the other to the C-terminal region. 5. The distribution of iron in human serum transferrin partially saturated with various iron donors was examined by electrophoresis in urea/polyacrylamide gels and the two possible monoferric forms were unequivocally identified. 6. The site designated A on human serum transferrin [Harris (1977) Biochemistry 16, 560-564] was assigned to the C-terminal region of the protein and the B site to the N-terminal region. 7. The distribution of iron on transferrin in human plasma was determined

Studies on the changes in protein fluorescence and enzymic activity of aspartate aminotransferase on binding of pyridoxal 5'-Phosphate

1. The a and ,B subforms of aspartate aminotransferase were purified from pig heart. 2. The a subform contained 2mol of pyridoxal 5'-phosphate. The apo-(a subform) could be fully reactived by combination with 2mol of cofactor. 3. The protein fluorescence of the apo- (a subform) decreased non-linearly with increase in enzyme activity and concentration of bound cofactor. 4. It is concluded that the enzyme activity/mol ofbound cofactor is largely independent of the number ofcofactors bound to the dimer. 5. The /Jsubformhad approximately half the specific enzyme activity of the a subform, and contained an average of one active pyridoxal 5'-phosphate molecule per molecule, which could be removed by glutamate, and another inactive cofactor which could only be removed with NaOH. 6. On recombination with pyridoxal 5'-phosphate the protein fluorescence of the apo-(fl subform) decreased linearly, showing that each dimeric enzyme molecule contained one active and one inactive bound cofactor. 7. The results are not consistent with a flip-flop mechanism for this enzyme

Chelator-facilitated removal of iron from transferrin: Relevance to combined chelation therapy

Current iron chelation therapy consists primarily of DFO (desferrioxamine), which has to be administered via intravenous infusion, together with deferiprone and deferasirox, which are orally-active chelators. These chelators, although effective at decreasing the iron load, are associated with a number of side effects. Grady suggested that the combined administration of a smaller bidentate chelator and a larger hexadentate chelator, such as DFO, would result in greater iron removal than either chelator alone [Grady, Bardoukas and Giardina (1998) Blood 92, 16b]. This in turn could lead to a decrease in the chelator dose required. To test this hypothesis, the rate of iron transfer from a range of bidentate HPO (hydroxypyridin-4-one) chelators to DFO was monitored. Spectroscopic methods were utilized to monitor the decrease in the concentration of the Fe–HPO complex. Having established that the shuttling of iron from the bidentate chelator to DFO does occur under clinically relevant concentrations of chelator, studies were undertaken to evaluate whether this mechanism of transfer would apply to iron removal from transferrin. Again, the simultaneous presence of both a bidentate chelator and DFO was found to enhance the rate of iron chelation from transferrin at clinically relevant chelator levels. Deferiprone was found to be particularly effective at ‘shuttling’ iron from transferrin to DFO, probably as a result of its small size and relative low affinity for iron compared with other analogous HPO chelators

The iron binding-sites of chicken ovotransferrin

We have shown previously that the EXAFS spectrum of diferric chicken ovotransferrin (Fe2COT) can only be adequately simulated assuming a split first shell co-ordination [1]. EXAFS and XANES spectra of Fe2COT measured in solution and as a freeze-dried powder provide evidence for perturbation of the iron-binding sites on freeze-drying which involves the loss of one of the long (~2.04 Å) first shell ligands (presumably water). Measurement of the XANES of the C-terminal monoferric COT and a C-terminal domain fragment suggests that the metal binding site remains largely unperturbed by the fragmentation process. The possibility of site interaction is briefly discussed.We gratefully acknowledge the SERC for financial support and provision of facilities

Homology modelling of transferrin-binding protein A from Neisseria meningitidis

Neisseria meningitidis, a causative agent of bacterial meningitis, obtains transferrin-bound iron by expressing two outer membrane located transferrin-binding proteins, TbpA and TbpB. TbpA is thought to be an integral outer membrane pore that facilitates iron uptake. Evidence suggests that TbpA is a useful antigen for inclusion in a vaccine effective against meningococcal disease, hence the identification of regions involved in ligand binding is of paramount importance to design strategies to block uptake of iron. The protein shares sequence and functional similarities to the Escherichia coli siderophore receptors FepA and FhuA, whose structures have been determined. These receptors are composed of two domains, a 22-stranded b-barrel and an N-terminal plug region that sits within the barrel and occludes the transmembrane pore. A three-dimensional TbpA model was constructed using FepA and FhuA structural templates, hydrophobicity analysis and homology modelling. TbpA was found to possess a similar architecture to the siderophore receptors. In addition to providing insights into the highly immunogenic nature of TbpA and allowing the prediction of potentially important ligandbinding epitopes, the model also reveals a narrow channel through its entire length. The relevance of this channel and the spatial arrangement of external loops, to the mechanism of iron translocation employed by TbpA is discussed

The importance of electron temperature in silicon-based terahertz quantum cascade lasers

Quantum cascade lasers (QCLs) are compact sources of coherent terahertz radiation. Although all existing QCLs use III-V compound semiconductors, silicon-based devices are highly desirable due to the high thermal conductivity and mature processing technology. We use a semiclassical rate-equation model to show that Ge/SiGe THz QCL active region gain is strongly enhanced by reducing the electron temperature. We present a bound-to-continuum QCL design employing L-valley intersubband transitions, using high Ge fraction barriers to reduce interface roughness scattering, and a low electric field to reduce the electron temperature. We predict a gain of similar to 50 cm(-1), which exceeds the calculated waveguide losses. (C) 2009 American Institute of Physics. [doi: 10.1063/1.3237177