Transferrin preparation

The expression of several genes is induced or repressed by hemopexin-mediated heme transport. Most of these are simple responses of the cell to the increased heme (or iron derived from heme) in the cell. For example, HO-1 is induced (15, 88), ferritin levels rise (14, 61, 89), the transferrin receptor is down-regulated (15), and hemopexin mRNA itself is induced (A. Smith, unpublished). However, MT-1 is also induced, apparently to prepare the cell for oxidative stress thus, in addition to sequestering heme in a low-spin, non-oxidatively active form, hemopexin also indirectly exerts antioxidant effects by inducing MT-1 (16, 61, 90). 

Iron(III)-pyrophosphate looks promising as an alternative to iron(III)-carbohydrate preparations for parenteral administration for treatment of anemia.Kinetics of removal of iron from transferrin (tf) by pyrophosphate (pp) were found to be biphasic under certain conditions, with the rapid first phase attributed to the formation of a pp—Fe—tf—CO intermediate.A later study of the kinetics of removal of iron from transferrin employed pyrophosphate and tripodal phosphonates such as nitrilotris(methylenephosphonic acid), N(CH2P03H2)3. For the tripodal ligands there are parallel first-order and saturation pathways, with the latter dominant (contrast the exclusively first-order reaction of ferritin with nitrilotriacetate) for pyrophosphate the paths are roughly equal in importance. The saturation kinetics suggest that tfiFe-phosphonate intermediates play an important role in the kinetics. 

In 1993, another immunoassay for the detection of monensin was developed but, unfortunately, was never applied to biological material (91). Quite recently a competitive ELISA and a compatible extraction procedure suitable for screening monensin in poultry liver samples was described (92). In this assay, a polyclonal antiserum raised against a monensin-transferrin conjugate and prepared via an acid anhydride intermediate (93) was used. Significant cross-reactivity with other polyethers commonly used by the broiler industry, such as maduramicin, lasalocid, salinomycin, and narasin, was not found. A detection limit of 3 ppb could be readily attained when liver samples were submitted to extraction with aqueous acetonitrile, partitioning between aqueous sodium hydroxide solution and a hexane-diethyl either mixture, evaporation of the organic phase, and reconstitution in ethanol/sodium acetate solution. 

Obtain a precast SDS-polyacrylamide slab gel or prepare one according to instructions in Experiment 4. The recommended gel is 12°/o acrylamide with a thickness of 0.75 mm. Protein samples are prepared as follows Purified proteins (transferrin, bovine serum albumin, a, -antitrypsin, a-lactalbumin from Experiment 4, and molecular weight standards) are supplied in Tris buffer, pH 6.8 solutions at a concentration of 1 mg/mL. Sera samples have been diluted and are ready for use. Prepare protein samples for electrophoresis in 0.5-mL microcentrifuge tubes with attached caps. Label the tubes from 1 to 5 as below or per your Instructor s directions. 

PDPH has been used in the preparation of immunotoxin conjugates (Zara et al., 1991). It has also been used to create a unique conjugate of NGF with an antibody directed against the transferrin receptor OX-26, which could traverse the blood-brain barrier (Friden et al., 1993). Labeling of antibody molecules with PDPH at oxidized polysaccharide sites followed by reduction to free the sulfhydryl has been used to form a technetium-99m complex for radiopharmaceutical use (Ranadive et al., 1993) (Chapter 8, Section 2.5). 

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). 

The methods that have been used in the preparation of the various transferrins include most of those generally used in the preparation of proteins. In addition, the particular properties of the iron complexes have been used for the development of adjunctive procedures. The iron complexes have solubilities that differ from those of the metal-free proteins, and the iron complexes are relatively stable proteins as compared to most other proteins. The stability of the iron complex can be used to denature and insolubilize preferentially other proteins present as contaminants. 

Schade and Caroline (116) found the serum transferrin primarily in human blood serum fraction IV—3,4 (according to Cohn s nomenclature). The transferrin was purified and concentrated in fraction IV—7 by further subfractionations (30, 125, 126). Crystallized transferrin was prepared by crystallization in low dielectric solvents under low temperatures at controlled ionic strength and pH (81, 86). 

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). 

Fiala (45) and Fiala and Burk (46) early postulated, by analogy from the visible absorption spectra of iron transferrin and the iron complex of aspergillic acid, that iron was bound in transferrins through a hydroxamic acid-CC>2 complex. This formulation is shown in Fig. 15. Fraenkel-Conrat (48), however, could find no evidence for hydroxylamido groups in chicken ovotransferrin. He also prepared and studied the properties of several hydroxylamido proteins by the chemical introduction of the hydroxylamido groups, and found that their properties were quite different from those of the transferrins. 

Liposomes can be targeted to the brain by exploiting receptor-mediated transcytosis systems. For example, a bi-functional PEG-linker has been used to couple anti-transferrin (0X26) receptor antibodies to one end of the PEG strands and liposomes at the other end of the PEG strands (Figure 13.6). Classically, immunoliposomes are prepared by attaching the MAb to the surface of the liposomes (see Section 5.3.1.3). However, this can lead to steric hindrance by the PEG strands with respect to antibody binding to the appropriate receptor. The use of the bifunctional PEG linker overcomes this problem. 

Figure 2. Undecoupled 13C NMR Fourier transform spectra of Co(III)-transferrin-CO3. (A) 13CO -labeled transferrin (B) no label (C) after adding H13COf to the preparation used for A. The line at 104 ppm is caused by 13C03 specifically bound to transferrin and is 14 Hz wide. The linewidth of free H13C03 at 96 ppm is 7 Hz (32).

Since malonate (Structure I) is able to fulfill the role of the anion in transferrin, it seemed reasonable to see whether spin-labeled derivatives of malonate could serve as probes of the active sites. Two such spin-labled derivatives were prepared and tentatively identified as having structures II (N-4-(2,2,6,6-tetramethylpiperidin-l-oxyl)malonamide) and III (N-4-(2,2,6,6-tetramethylpiperidin-l-oxyl)malonate). Similar results were obtained with each (Figure 3). Upon mixing Fe(III), transferrin, and II at low pH, and then raising the pH to near-neutrality with C02-free ammonia, the characteristic orange-red color of the ternary Fe-transferrin-anion complex is promptly displayed. However, the anticipated EPR signal of the nitroxide spin-label is not observed, presumably because it is broadened beyond detectability by its proximity... 

GHz Fe(III)-transferrin-ll complex at room temperature. The 5 X 10 4M transferrin solution was about 90% saturated with Fe(III) and nitroxyl-labeled malonate (compound II). (B) After the preparation was made 5 X 10 3M in bicarbonate (49). 

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