Transferrins kinetics

FIG. 15 Cellular entry and intracellular kinetics of the cationic lipid-DNA complexes. Cationic lipid-DOPE liposomes form electrostatic complexes with DNA, and, in this case, also transferrin (Tf) is incorporated. Cellular uptake by endoc5dosis and endosomal acidification can be blocked with cytochaiasin B and bafilomycin Aj, respectively. DNA is proposed to be released at the level of endosomal wall after fusion of the carrier lipids with endosomal bilayer. This process is facilitated by the formation of inverted hexagonal DOPE phase as illustrated in the lower corner on the right. After its release to the C5doplasm DNA may enter the nucleus. (From Ref. 253. By permission of Nature Publishing Group.)... 

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. 

Fe(III) displacement of Al(III), Ga(III), or In(III) from their respective complexes with these tripodal ligands, have been determined. The M(III)-by-Fe(III) displacement processes are controlled by the ease of dissociation of Al(III), Ga(III), or In(III) Fe(III) may in turn be displaced from these complexes by edta (removal from the two non-equivalent sites gives rise to an appropriate kinetic pattern) (343). Kinetics and mechanism of a catalytic chloride ion effect on the dissociation of model siderophore-hydroxamate iron(III) complexes chloride and, to lesser extents, bromide and nitrate, catalyze ligand dissociation through transient coordination of the added anion to the iron (344). A catechol derivative of desferrioxamine has been found to remove iron from transferrin about 100 times faster than desferrioxamine itself it forms a significantly more stable product with Fe3+ (345). 

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. 

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. 

An order of effectiveness has been established and a mechanism proposed for the removal of iron from ferritin by several 3-hydroxy-4-pyridinone chelators. The removal of iron from ferritin is, as one would expect, considerably slower than from calcein or doxorubicin (cf. above) or from transferrin. Rate constants are between 1.5 x 10 s and 7.5 x 10 s for removal of iron from ferritin by a series of hexadentate ligands each consisting of three substituted A-hydroxypyrimidinone or A-hydroxypyrazinone units, the rate decreasing with increasing substituent bulk. The slowest rate approximates to that for removal of iron from ferritin by desferrioxamine. The influence of chirality on the kinetic barrier provides insight into the detailed mechanism of removal in these systems.Slow removal of iron from ferritin by chelators should be contrasted with rapid reductive removal. ... 

Steric factors govern the rate of iron transfer. Using Fe-EDTA and transferrin, it has been demonstrated by kinetic, spectral, and EPR... 

Ciechanover, A., Schwartz, A. L., Dautry-Varsat, A., and Lodish, H. F. Kinetics of internalization and recycling of transferrin and the transferrin receptor in a human hepatoma cell line. Effect of lysosomotropic agents. J. Biol. Chem. 258 9681-969, 1983. 

Investigations of the kinetics of the reaction of these new siderophores with iron-saturated transferrin showed a rapid formation of a ternary complex with transferrin, followed by a slow step in which the ferric siderophore was released from the apoprotein. Weitl et al.257 have evaluated the ferric-chelating properties of several of these siderophores and found the following order of effectiveness for removing iron from transferrin enterobactin > MECAMS > MECAM > LICAMS > DFOA > TRIM-CAMS. 

Marques HM. 1991. Kinetics of the release of aluminum from human serum dialuminum transferrin to citrate. J Inorg Biochem 41 187-193. 

Treating ovotransferrin and human serum transferrin with 170-400 molar excess of ethoxyformic anhydride resulted in complete ethoxy-formylation of histidines with complete loss in iron-binding activity (33). The binding of each iron (two iron-binding sites per protein molecule) protected two histidines from ethoxyformylation, and in both cases the proteins remained completely active. These results plus kinetic analyses of the inactivations indicated two essential histidines in each binding site. Ethoxyformic anhydride also may react with amino groups. 

The two sites also differ in their pH stability towards iron release. Experiments on serum transferrin showed that one site loses iron at a pH near 6.0, and the other at a pH nearer 5.0 (203, 204), giving a distinctly biphasic pH-induced release profile (Fig. 28). The acid-stable A site was later shown to be the C-terminal site (202). It is this differential response to pH, together with kinetic effects (below), that enables N-terminal and C-terminal monoferric transferrins to be prepared (200). Although the N-terminal site is more labile, both kinetically and to acid, the reasons are not necessarily the same the acid stability may depend on the protonation of specific residues (Section V.B) and is likely to differ somewhat from one transferrin to another in response to sequence changes. The biphasic acid-induced release of iron seen for transferrin is not shared by lactoferrin. Although biphasic release from lactoferrin, in the presence at EDTA, has been reported (205), under most conditions both sites release iron essentially together at a pH(2.5-4.0) several units lower than that for transferrin (Fig. 28). 

The two sites (in transferrin, at least) also show differences in iron loading behaviour. In vitro, when Fe3+ is added as a chelate complex, there are differences in which site is preferentially loaded, depending on the nature of the chelate ligand these differences are apparently kinetically determined and differ from one transferrin to another (17). 

In vivo uptake of iron by transferrins usually involves its addition as a ferric-chelate complex, to prevent hydrolytic attack on the ferric ion (211). Complexes such as ferric citrate and ferric nitrilotriacetate are commonly used. Under these conditions, kinetic schemes for the uptake of iron by transferrins have identified five steps in the formation of the specific metal-anion-transferrin ternary complex (120). These may be summarized as follows. 

Many studies have noted weak cooperativity between the sites during iron release (3). One recent analysis used mixed-metal transferrins, with kinetically inert Co3+ in one site and Fe3+ in the other (221,224). With pyrophosphate, release of iron from the C-site was accelerated by the presence of a metal in the N-site, but no corresponding effect was seen for iron release from the N-site. The cooperative effects were also weaker and somewhat different for different chelators (221). 

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