Transferrin analysis

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. 

In humans the clearance rate of Hb is higher than that of HpHb (L4, Lll). Murray et al. (M6) found this also to hold for rabbits and, by studying the elimination in nephrectomized animals, they also proved that the difference was not due to urinary loss of Hb. Analysis of the organs proved that the HpHb complex and Hb were assimilated mainly in the liver and were catabolized with an early reappearance of the iron as transferrin iron within 30 minutes. The free Hb accumulated also in the tubular cells of the kidneys. No data have been published suggesting that the spleen is of any appreciable importance in this respect. No typical exponential clearance of the HpHb complex from plasma was observed (L10, Lll) in the first few experiments. Lathem and Worley (L4) found that HpHb disappeared at a simple exponential rate in 5... 

Kinetic studies were attempted at 4, 15, 25 and 37 °C, but the colloids tended to aggregate at all temperatures above 4°C. Four models were used to determine the binding mechanisms from the kinetic data. A detailed analysis of binding at 4 °C, was made. Models were set up involving one or two surface sites which also satisfied the overall kinetics but the analyses were not definitive. Although it was demonstrated that the cells were capable of endocytosis of fluorescence-conjugated transferrin, there was no evidence for the endocytosis of the cationic colloids. 

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. 

Long values of Tig, consistently with expectations, were obtained for some VO-protein compounds. Analysis of the NMRD profile of bis-oxovanadium(IV) transferrin (59), for instance, provides a value for Xg of... 

On release of iron, transferrin undergoes a major conformational change, characterized by a hydrodynamically less compact structure. Crystallographic analysis of apolactoferrin reveals that the amino-... 

Fig. 4.5.2 Actual strategies for CDG diagnosis. Initial investigations on CDG patients are routinely carried out by isoelectric focusing (IEF) of serum transferrin. With a CDG type I pattern, subsequent analysis should imply determination of phosphomannomutase (PMM) and phos-phomannose isomerase (PMI) activities. Further studies, like analysis of the lipid-linked- and protein-bound-oligosaccharides, determination of enzyme or sugar transporter activities and molecular biology studies often have to be performed in more specialised laboratories. HPLC High-performance liquid chromatography, TLC thin-layer chromatography...

Data analysis is carried out by comparing IEF patterns of controls and defined CDG types with patients in suspicion of CDG. IEF of transferrin from controls show predominantly the tetrasialo form of the protein, whereas in case of CDG-I patients additional di- and asialo bands appear (Fig. 4.5.3). 

In the case of unclear transferrin patterns obtained from IEF analysis of blood samples derived from Guthrie cards, the analysis should be repeated with serum. 

Data analysis is carried out by comparing IEF patterns from patients suspected of having a CDG with healthy controls and already defined CDG types. Following neuraminidase treatment, controls and CDG patients will predominantly present with the asialo form of transferrin. In the case of mutations that affect the protein backbone of transferrin, additional bands appear (Fig. 4.5.4). 

Metabolic labelling studies on LLO with [2-3H] mannose are carried out in fibroblasts of patients who present with a characteristic CDG-type IIEF transferrin pattern but normal PMM and PMI activities, thereby excluding CDG-Ia and CDG-Ib. Investigations require the extraction and analysis of LLO by HPLC and TLC. 

Fig. 4.5.8 Examples for structures and molecular masses of 2-aminobenzamide (2-AB)-labelled oligosaccharide moieties derived from serum transferrin. Values below the oligosaccharide structures indicate the expected masses (in Da) by matrix-assisted laser desorption ionisation - time of flight analysis.

Fig. 4.5.9 HPLC and mass-spectromet-ric analysis of transferrin-linked oligosaccharides. Transferrin is purified from sera of a control and the index CDG-IId patient. Oligosaccharides are released by N-glycosidase F digestion and subsequently analysed by HPLC. Peak fractions of the control and the patient are further investigated by mass spectrometry and are compared to oligosaccharide standards. Values above the HPLC peaks indicate the detected masses...

Iron for biosynthesis is transported through the bloodstream by the protein transferrin. The following procedure measures the Fe content of transferrin 10 This analysis requires only about 1 p,g for an accuracy of 2-5%. Human blood usually contains about 45 vol% cells and 55 vol% plasma (liquid). If blood is collected without an anticoagulant, the blood clots, and the liquid that remains is called serum. Serum normally contains about 1 pg of Fe/mL attached to transferrin. 

The H NMRD profile of the diferric transferrin solution (Fig. 5.5) [3] is also instructive for the case of a macromolecule containing a Fe(III) atom. The profile shows four inflections the first is ascribed to the cos dispersion, the second one to the transition from the dominant ZFS limit to the dominant Zeeman limit (see Section 3.7.1), the following increase is due to the field dependent electron relaxation time (see Section 3.7.2) and finally the coj dispersion appears. The best fit analysis provides the presence of a rhombic ZFS with D = 0.2, E/D = 1 /3, in accordance with EPR spectra [9]. The analysis suggests that two sets of electron relaxation times must be considered, in the range 0.3-1 x 10 9 s. In fact, Eqs. (3.11) and (3.12) are inadequate to describe the field dependence of the electron relaxation over the whole range of frequencies due to the presence of static ZFS [10]. 

Using PMFs for identification allows crossspecies identification of highly conserved proteins, suggested by Liska and Shevchenko (2003). Proteins such as regucalcin, transferrin and enolase 1 have been identified from ovine host bile using sequences from bovine sources (Fig. 17.2) in the analysis of the sheep-liver... 

Bergen HR, Lacey JM, O Brien JF, et al. Online single-step analysis of blood proteins The transferrin story. Anal. Biochem. (2001) 296 122-129. 

In 1970, Eckman et al. devised the automation of a quantitative immunochemical analysis of transferrin (El). In this automated flow system, diluted samples were allowed to react with antitransferrin antiserum serially and the degree of light scattering of the resulting turbidity was measured in the fluorometer, used as a nephelometer. The optimal conditions for nephelometry were extensively studied. Subsequently, Buffone reported... 

---