Transferrin

Abstract

The interaction of apolactoferrin with hydrogen carbonate (bicarbonate) has been investigated in the pH range 6.5-9.2. In the absence of bicarbonate apolactoferrin loses a single proton with pK,, of 8.10. This proton loss is independent of the interaction with the synergistic anion. The C-site of apolactoferrin interacts with bicarbonate with a very low affinity (K;' = 3.2 M-'). This process is accompanied by a proton loss, which is probably provided by the bicarbonate in interaction with the protein. This proton loss can possibly be the result of a shift in the proton dissociation constant, pK,, of the bicarbonate/ carbonate acid/base equilibrium, which would decrease from pK, 10.35 to pK,, 6.90 in the bicarbonatelactoferrin adduct. The N-site of the protein interacts with bicarbonate with an extremely low affinity, which excludes the presence of the N-site-synergistic anion adduct in neutral physiological media. Contrary to serum transferrin, the concentration of the apolactoferrin in interaction with bicarbonate is pH dependent. Between pH 7.4 and pH 9 with [HCO,] about 20 mM, the concentration of the serum transferrin-bicarbonate adduct is always about 30 %, whereas that of the apolactoferrin-synergistic anion adduct varies from 25% at pH 7.5 to 90% at pH 9. This implies that, despite an affinity for bicarbonate two orders of magnitude lower than that of serum transferrin, lactoferrin interacts better with the synergistic anion. This can be explained by the possible interaction of lactoferrin with carbonate in neutral media, whereas transferrin only interacts with bicarbonate.Keywords; transferrin ; lactoferrin; synergistic anion ; iron metabolism; iron transport.The transferrin family constitutes the most important irontransport system in vertebrates and in some invertebrates, mainly worms and insects (Aisen, 1989;Kurama et al., 1995). These proteins transport iron from the biological fluid into the cytoplasm via the plasma membrane by receptor-mediated endocytosis. With the exception of melanotransferrin, which is overexpressed in melanocytes, the other transferrins of the family are soluble glycoproteins (Baker and Lindley, 1992). These are represented by serum transferrin, responsible for iron transport in mammals, egg white ovotransferrin and milk lactoferrin, which is also present in tears, genital secretion, seminal fluid, sweat, etc. (Aisen, 1989). In addition to their iron-transport function, some transferrins, such as lactoferrins, are involved in the immune response and are bactericidal and bacteriostatic (FurmanCorrespondence to J

Transferrins

“…This implies that the interaction with bicarbonate is independent of the state of protonation of the ovotransferrin as reported for serum transferrin (Bellounis et al, 1996). We may also assume to a first approximation, as in the case of serum transferrin and lactoferrin, that the interaction with bicarbonate occurs with both sites, with however, one of the sites (presumably the C-site) presenting a much greater affinity for the synergistic anion (Bellounis et al, 1996;Pakdaman and El Hage Chahine, 1997). We can therefore write for the C-site :…”

“…Protein purification. Ovotransferrin (Sigma) was purified and its purity and iron load were checked by spectrophotometric analysis and by urea-polyacrylamide gel electrophoresis, as previously described (Makey and Seal, 1976;Pakdaman and El Hage Chahine, 1997). All our experiments were performed with the purified apo-ovotransferrin.…”

“…Two were with [HCO Ϫ 3 ] varying from 0 to 40 mM at fixed pH 7.3 and pH 9.0 where the major species present in the medium are OTH m in acidic and OTH mϪ2 in basic media. Experimental data obeyed for each pH value Eqn (7) (Pakdaman and El Hage Chahine, 1997).…”

“…In each lobe, iron is co-ordinated to the four lateral chains of two tyrosines, one histidine, one aspartate, and a carbonate or bicarbonate adjacent to an arginine (Bailey et al, 1988;Anderson et al, 1989;Zuccola, 1992;Kurokawa et al, 1995;Rawas et al, 1996;Moore et al, 1997). This carbonate constitutes the synergistic anion which gives these proteins affinities for iron in the 10 20 M Ϫ1 range (Aisen, 1989;Bellounis et al, 1996;Pakdaman and El Hage Chahine, 1997). However, although soluble transferrins are very similar and present a high degree of similarity in their amino acid sequences (Baldwin, 1993), some differences exist in their structures (Anderson et al, 1989;Kurokawa et al, 1995;Rawas et al, 1996;Moore et al, 1997).…”

Transferrin as a Metal Ion Mediator

“…128,[143][144][145] Apo-lactoferrin interacts strongly with this bicarbonate, possibly due to the binding of carbonate to apo-lactoferrin instead of bicarbonate as for apo-transferrin. Evidence for an intermediate anion-metal-chelate-transferrin complex has been obtained from studies using Fe III -acetohydroxamate in iron uptake experiments.…”