Transferrin, human serum

Kilar, E and Hjerten,S., Fast and high resolution analysis of human serum transferrin by high performance isoelectric focusing in capillaries, Electrophoresis, 10, 23, 1989. 

Kilar, F. and Fanali, S., Separation of tryptophan-derivative enantiomers with iron-free human serum transferrin by capillary zone electrophoresis, Electrophoresis, 16, 1510, 1995. 

Determination of the amino-acid sequence of human serum transferrin (MacGillivray et ah, 1983) and of human lactoferrin (Metz-Boutique etal., 1984) revealed an internal two-fold sequence repeat. The amino-terminal half has approximately 40 % sequence identity with the carboxyl-terminal half. Similar results have subsequently been found for a number of other transferrins (Baldwin,... 

A recently obtained high resolution structure of two crystal forms of the N-lobe of human serum transferrin (at 0.16 and 0.18 nm resolution) shows disorder at the iron-binding sites (MacGillivray et ah, 1998). Model building and refinement show... 

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.)... 

Schewale, J.G., and Brew, K. (1982) Effects of Fe3+ binding on the microenvironments of individual amino groups in human serum transferrin as determined by different kinetic labeling. /. Biol. Chem. 257, 9406. 

Mason, A.B., Halbrooks, P.J., James, N.G., Connolly, S.A., Larouche, J.R., Smith, V.C., MacGillivray, R.T.A. and Chasteen, N.D. (2005) Mutational analysis of C-lobe ligands of human serum Transferrin insights into the mechanism of iron release, Biochemistry, 44, 8013-8021. 

An interesting application of CZE was the study of the interaction of human serum transferrin and fluorescein isothiocyanate (FITC). Measurements were carried out in an uncoated fused-silica capillary (total length 59 cm 75 pm i.d. effective lengths for... 

T. Konecsni and F. Kilar, Monitoring of the conjugation reaction between human serum transferrin and fluorescein isothiocyanate by capillary electrophoresis. J. ChromatogrA, 1051 (2004) 135-139. 

Bacterial hosts are inappropriate choices for expression of proteins such as the blue copper proteins stellacyanin, laccase, and ceruloplasmin which are extensively glycosylated. In these cases, it may be necessary to employ tissue cultures of appropriate origin to obtain the native protein. In this regard, the amino-terminal half of human serum transferrin, which lacks carbohydrate, has been expressed in high yield in baby hamster kidney cells by Funk et al. [13], while the glycosylated carboxyl-terminus has proved to be more problematic [103]. 

F Kilar, S Hjerten. Unfolding of human serum transferrin in urea studied by high-performance capillary electrophoresis. J. Chromatogr. 638 269-276... 

F Kilar. Stereoselective interaction of drug enantiomers with human serum transferrin in capillary zone electrophoresis. Electrophoresis 17 1950-1953, 1996. 

The significance of the two sites in transferrin has been much discussed. The sites are distinguishable spectroscopically and have different affinities for iron, which may be dependent on the anion used. The two sites release iron at different rates in a pH-dependent manner. The site in the C-terminal half of human serum transferrin (once designated the A site) retains its iron at pH 6.0 and so is the acid-stable site. The site on the N-terminal half is the acid-labile site. 

FIG. 34.—Molecular Model of Diantennary Glycan of Human-serum Transferrin. [A, Y Conformation14 B, T Conformation. 4 Numbers correspond to the numbering used in Table II (see fundamental structure).]... 

Kubal G, Mason AB, Sadler PJ, et al. 1992. Uptake of Al 3+into the N-lobe of human serum transferrin. Biochem J 285 711-714. 

Kay, C. W. M., Mkami, H. E., Cammack, R., and Evans, R. W. (2007). Pulsed ELDOR determination of the intramolecular distance between the metal binding sites in dicupric human serum transferrin and lactoferrin. J. Am. Chem. Soc. 129, 4868. 

Guo M, Sun H, McArdle HJ, Gambling L, Sadler PJ (2000) TiIV uptake and release by human serum transferrin and recognition of TiIV-transferrin by cancer cells understanding the mechanism of action of the anticancer drug titanocene dichloride. Biochemistry 39 ... 

Since these earlier studies a variety of different methods and techniques have appeared for purifications of the serum transferrins. Precipitation of human serum transferrin by rivanol appears to have been used widely as an initial purification step (17, 18, 80, 107). A method for the preparation of large quantities of human serum transferrin was proposed by Inman et al. (69). The Inman method employed solvent and salt fractionation and cellulose ion exchange chromatography. A number of other workers have used cellulose ion exchange chromatography in combination with other procedures, such as electrophoresis (14, 22, 57, 105, 112, 137). 

Human serum transferrin has been prepared in the laboratories of the University of California, Davis, from Cohn fraction IV—7 obtained from a commercial company (Cutter Laboratories, Berkeley, California). Such fractions are, of course, from plasma of a large number of individuals and may have been exposed to different treatments. For example, some of these fractions have been exposed to a heating step to inactivate viruses. This type of human material, which is the usual type used for large scale preparations, certainly contains different molecular forms, because of genetic differences, as well as some artifactual materials. The procedures employed molecular filtration on Sephadex columns and sequential ion exchange chromatography on anion and cation cellulose exchangers (22, 137). 

Table 2. Amino acid composition of human serum transferrin ...

As compared to the information on the carbohydrate contents of human serum transferrin, much less information is available on lacto-transferrin. In general, however, the available values for lactotransferrin are more similar to those for serum transferrin than to those for ovotrans-ferrin. 

With but few exceptions, definitive studies on the properties of the metal complexes have been done with human serum transferrin and chicken ovotransferrin, the majority having been done with the chicken ovotransferrin. Many of the properties of the metal complexes are very similar to, if not identical with, those of the metal-free proteins but also there are several rather distinctive differences. 

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. 

Fig. 5. Absorption spectra for colored complexes of human serum transferrins. (Biochemistry 2, 1335 [1963]).

The importance of carbon dioxide in the formation of the iron complexes of the human serum transferrin was shown early by Fiala and Burk (46) and Schade et al. 117). Warner and Weber (133), by an indirect but exquisite method, proved the participation of CO2 in the formation of the complex, and also that it was in the form of bicarbonate or carbonate. This was done through experiments in which the iron complex was formed in the absence of CO2, or by the addition of small amounts, of gaseous CO2, and noting the red color of the complex was formed only very slowly. However, when the enzyme carbonic anhydrase was added, together with the CO2, the red color of the complex appeared rapidly. In addition the same workers showed by the use of C1402 that one mole of CO2 was bound per mole of iron bound. 

All the transferrins studied have been found to bind two gram atoms of metal ion per mole of protein. The metal ions are dissociated in acid solution but are stably bound to pH 9 or 10, depending upon the ion. The relative strengths of the ovotransferrin complex have been shown to be Fe3+ ) Cu2+ ) Zn2+ (Fraenkel-Conrat and Feeney, (49) Warner and Weber, (133)). Inman (68) showed by displacement studies the following order of binding for human serum transferrin and ovotransferrin Co3+ ) Fe3+ ) Mna+ ) Cu2+. 

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