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Differences in the protein fluorescence of the two iron(lI1)-binding sites of ovotransferrin
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
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Protein fluorescence of nicotinamide nucleotide-dependent dehydrogenases
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