Ligand variation in the transferrin family: the crystal structure of the H249Q mutant of the human transferrin N-lobe as a model for iron binding in insect transferrins

Proteins of the transferrin (Tf) family play a central role in iron homeostasis in vertebrates. In vertebrate Tfs, the four iron-binding ligands, 1 Asp, 2 Tyr, and 1 His, are invariant in both lobes of these bilobal proteins. In contrast, there are striking variations in the Tfs that have been characterized from insect species; in three of them, sequence changes in the C-lobe binding site render it nonfunctional, and in all of them the His ligand in the N-lobe site is changed to Gln. Surprisingly, mutagenesis of the histidine ligand, His249, to glutamine in the N-lobe half-molecule of human Tf (hTf/2N) shows that iron binding is destabilized and suggests that Gln249 does not bind to iron. We have determined the crystal structure of the H249Q mutant of hTf/2N and refined it at 1.85 A resolution (R = 0.221, R(free) = 0.246). The structure reveals that Gln249 does coordinate to iron, albeit with a lengthened Fe-Oepsilon1 bond of 2.34 A. In every other respect, the protein structure is unchanged from wild-type. Examination of insect Tf sequences shows that the K206.K296 dilysine pair, which aids iron release from the N-lobes of vertebrate Tfs, is not present in the insect proteins. We conclude that substitution of Gln for His does destabilize iron binding, but in the insect Tfs this is compensated by the loss of the dilysine interaction. The combination of a His ligand with the dilysine pair in vertebrate Tfs may have been a later evolutionary development that gives more sophisticated pH-mediated control of iron release from the N-lobe of transferrins.     

Production of transferrin receptors by Histophilus ovis: three of five strains require two signals

Five strains of Histophilus ovis (9L, 642A, 714, 5688T, and 3384Y) were investigated with respect to iron acquisition. All strains used ovine, bovine, and goat transferrins (Tfs), but not porcine or human Tfs, as iron sources for growth. In solid phase binding assays, total membranes from only two (9L and 642A) of the five strains, grown under iron-restricted conditions, were able to bind Tfs (ovine, bovine, and goat, but not porcine or human). However, when the organisms were grown under iron-restricted conditions in the presence of bovine transferrin (Tf), total membranes from all strains exhibited Tf binding (as above); competition experiments demonstrated that all three Tfs (ovine, bovine, and goat) were bound by the same receptor(s). Membranes from organisms grown under iron-replete conditions in the presence or absence of bovine Tf failed to bind any of the test Tfs. An affinity-isolation procedure allowed the isolation of two putative Tf-binding polypeptides (78 and 66 kDa) from total membranes of strains 9L and 642A grown under iron-restricted conditions, and from membranes of all strains if the growth medium also contained Tf. It is concluded that all strains tested acquire Tf-bound iron by means of siderophore-independent mechanisms involving surface receptors analogous to the Tf-binding proteins (TbpA and TbpB) found in comparable organisms; although iron restriction alone is sufficient to promote the expression of these proteins by strains 9L and 642A, their production by strains 714, 5688T, and 3384Y appears to require two signals, iron restriction and the presence of Tf.     

Class specificity of transferrin as a muscle trophic factor

The specificity of transferrin (Tf) in its exertion of a growth-promoting effect on myogenic cells was examined using serum Tfs from chick, dove, goose, turkey, bovine, horse, rabbit, rat, and swine and primary myogenic cells from chick, duck, quail, rabbit, and rat, and rat L6 cells. Avian Tfs were effective on avian cells but not on mammalian cells, while mammalian Tfs were effective on mammalian cells but not on avian cells. Dove and bovine Tfs were exceptional in that they were effective on some class-heterologous cells at higher concentrations and less so or completely ineffective on some class-homologous cells. Despite these exceptions, however, the relationship between Tfs and cells can be summarized as a class specificity. To exert the growth-promoting effect, it is prerequisite for Tf to bind its specific receptor on the cell surface. Using quail and L6 cells, we found that the binding of 125I-labeled chick and rat Tfs to the respective receptors of quail and L6 myoblasts was competitively inhibited by other kinds of effective Tfs, but not by ineffective ones. We conclude that the class specificity in myotrophic activity of Tf is due to the affinity between Tf and Tf receptor.     

Recombinant human transferrin: beyond iron binding and transport

Iron is indispensible for life and essential for such processes as oxygen transport, electron transfer and DNA synthesis. Transferrin (Tf) is a ubiquitous protein with a central role in iron transport and metabolism. There is evidence, however, that Tf has many other biological roles in addition to its primary function of facilitating iron transport and metabolism, such as its profound effect on mammalian cell growth and productivity. The multiple functions of Tf can be exploited to develop many novel applications. Indeed, over the past several years, considerable efforts have been directed towards exploring human serum Tf (hTf), especially the use of recombinant native hTf and recombinant Tf fusion proteins, for various applications within biotechnology and medicine. Here, we review some of the remarkable progress that has been made towards the application of hTf in these diverse areas and discuss some of the exciting future prospects for hTf.     

Rat aglycotransferrin and human monoglycotransferrin: production and metabolic properties

Rat transferrin (rTf), containing one complex glycan, and human transferrin (hTf), containing two complex glycans, were treated with peptide:N-glycosidase F (PNGase) under nondenaturing conditions. Apo rTf with a nonfucosylated standard biantennary glycan, but not its diferric counterpart, yielded satisfactory amounts (approximately 55% in 7 h) of aglyco Tf (AgTf). The presence of a chitobiose core fucose reduced yields to approximately 30%, whereas an additional NeuAc on the GlcNAc in the Man(alpha 1-3) branch had no adverse effect. Triton X-100 impaired deglycosylation. The main product (approximately 65%) obtained from apo hTf was monoglyco Tf (MgTf). Analysis of the cyanogen bromide fragments of MgTf revealed that PNGase did not discriminate between the two glycosylation sites of hTf. A negligible portion (2-4%) of AgTf, that was also obtained during the reaction, probably resulted from PNGase action on denatured hTf molecules. Modified Tfs were separated by affinity chromatography, radiolabeled, mixed with another preparation of interest, and injected in rats. Total-body radiation measurements showed that the half-life of rat AgTf was 19-20% shorter than that of rTf but 9% longer than that of asialo Tf. This suggests that close to 76% of the change in the degradative rate observed after desialylating rTf is referable to charge loss rather than the exposure of Gal residues. Human MgTf was catabolized by rats 8-9% faster than the parent protein, while human AgTf behaved in vivo like a denatured protein. It is concluded that sialyl carboxyls are a codeterminant of the normal lifetime of transferrins.     

Binding affinity of aluminium to human serum transferrin and effects of carbohydrate chain modification as studied by HPLC/high-resolution ICP-MS--speciation of aluminium in human serum

Aluminium (Al) in the blood is bound to transferrin (Tf), a glycoprotein of about 80kDa that is characterized by its need for a synergistic anion. In this focused review, the binding affinity of Al to Tf is surveyed in the context of our recent studies using on-line high-performance liquid chromatography/high-resolution inductively coupled plasma mass spectrometry (HPLC/HR-ICP-MS). Al in human serum without any in vitro Al-spikes was present in a form bound to the N-lobe site of Tf. The influences of sialic acid in the carbohydrate chain of human serum Tf (hTf) were studied using asialo-hTf, obtained by treatment with sialidase. The binding affinity of Fe was similar between asialo-hTf and native-hTf, while that of Al for asialo-hTf was larger than that for native-hTf, especially in the presence of oxalate, a synergistic anion. The above findings are discussed in relation to diseases in which the serum concentrations of carbohydrate-deficient Tf and oxalate are augmented.     

Steric and allosteric factors prevent simultaneous binding of transferrin-binding proteins A and B to transferrin

The ability to acquire iron directly from host Tf (transferrin) is an adaptation common to important bacterial pathogens belonging to the Pasteurellaceae, Moraxellaceae and Neisseriaceae families. A surface receptor comprising an integral outer membrane protein, TbpA (Tf-binding protein A), and a surface-exposed lipoprotein, TbpB (Tf-binding protein B), mediates the iron acquisition process. TbpB is thought to extend from the cell surface for capture of Tf to initiate the process and deliver Tf to TbpA. TbpA functions as a gated channel for the passage of iron into the periplasm. In the present study we have mapped the effect of TbpA from Actinobacillus pleuropneumoniae on pTf (porcine Tf) using H/DX-MS (hydrogen/deuterium exchange coupled to MS) and compare it with a previously determined binding site for TbpB. The proposed TbpA footprint is adjacent to and potentially overlapping the TbpB-binding site, and induces a structural instability in the TbpB site. This suggests that simultaneous binding to pTf by both receptors would be hindered. We demonstrate that a recombinant TbpB lacking a portion of its anchor peptide is unable to form a stable ternary TbpA-pTf-TbpB complex. This truncated TbpB does not bind to a preformed Tf-TbpA complex, and TbpA removes pTf from a preformed Tf-TbpB complex. Thus the results of the present study support a model whereby TbpB 'hands-off' pTf to TbpA, which completes the iron removal and transport process.     

Effects of sialic acid residues of transferrin on the binding with aluminum and iron studied by HPLC/high-resolution ICP-MS

Transferrins (Tfs) are glycoproteins with carbohydrate chains in the C-lobe. Carbohydrate-deficient Tfs (CDTs) with fewer sialic acids increased in several diseases. In this study, the affinity of metals (Al and Fe) to Tfs was compared between native- and asialo-Tf by on-line high-performance liquid chromatography/high-resolution inductively coupled plasma mass spectrometry, to clarify whether the presence of sialic acids influences the metal binding. Fe added as Fe-citrate in the presence of bicarbonate preferred the N-lobe site and the binding affinity was similar between native- and asialo-Tfs. Al-citrate added at Al/Tf = 1 also preferred the N-lobe site, while the binding affinity was higher to asialo-Tf than to native-Tf. In Al-oxalate addition, the affinity to the N-lobe site of both Tfs increased further. In the absence of bicarbonate, Al-oxalate showed a preference for the C-lobe site in native-Tf and comparable affinity to both lobes in asialo-Tf. In asialo-Tf, Al2-Tf was the largest peak even at Al/Tf = 1. Thus, the lack of sialic acid in glycans and the presence of oxalate enhanced the binding affinity of Al to Tf. Therefore, it was suggested that the binding affinity of Al in patients with CDTs may be enhanced.     

Indispensability of Iron-bound Chick Transferrin for Chick Myogenesis in Vitro

Myotrophic activity of highly purified chick transferrins (Tfs) to chick primary myogenic cells has been studied in a culture medium containing horse serum. Iron-binding to Tfs is indispensable for the activity. The removal of iron from Tfs gives rise to a complete loss of the activity and it is restored by iron-rebinding depending on the amount of bound iron. This result, combined with other physicochemical and immunological data, strongly, confirms that the myotrophic activity is exerted by the Tfs themselves, not by a contaminating material(s). It has been found that culture medium containing horse Tf which seems inadequate for the study of the biological effects of Tfs is, however, suitable for studies on chick Tfs, since horse Tf is inactive in promoting chick myogenesis. Terminal sialic acid residues are unrelated to myotrophic activity since Tfs with different numbers of residues (0, 1, and 2 moles/Tf molecule) are comprable in their activities. The mechanism of Tf action on cells and contradictions among previous papers as to the requirement of Tf for cell growth have been discussed from the viewpoint of an iron-donor with class-specificity.     

Phylogenetical and ontogenetical studies on the molecular weight heterogeneity of bovine serum transferrin

Antitransferrin (Tf) rabbit serum was highly specific: it reacted with Tfs of ruminants, such as European breeds and Zebu breeds of cattle, Bali cattle, banteng, swamp and river types of water buffalo, anoa, goat, sheep, deer, antelope, camel, and giraffe, but did not react with serum of other non-ruminant species, such as pig, wild boar, hippopotamus, horse, rabbit, rat, chicken, etc. Electrophoresis of Tf and immunoglobulin G (IgG) complexes was carried out using sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). Within ruminants, the following species showed two Tf molecules on SDS-PAGE; European and Zebu cattle, Bali cattle, banteng, two types of water buffalo, and two species of anoa. Other ruminants, sheep, goat, deer, antelope, camel, and giraffe, etc., showed only one Tf molecule. The Tf heterogeneity in molecular weight was, thus, restricted to Bos, Bubalus, and Anoa. The molecular weight of Tf of water buffalo was slightly larger than that of cattle on the gel. The peptide pattern from cyanogen bromide cleavage of Tf of the water buffalo differed clearly from that of cattle. Fetal Tf showed only one molecule during development, but a newborn calf has two Tf molecules, (one large and one small) within 18 hr after birth. We suggest, therefore, that the small molecules formed during the last month of gestation. The peptide patterns of adult and fetal Tfs cleaved by cyanogen bromide differed with regard to the two large peptides; fetal Tf, lacking the second-largest peptide, had twice the amount of the largest peptide compared with adult Tf. From these results, we suggest that a change in peptide sequence occurs from the last month of gestation, when the largest peptide is degraded to the second largest. However, a Tf-like protein detected in the liver microsomal fraction has only one molecular size, both in adult and in fetal livers.     

Conformational stability of porcine serum transferrin.

The conformation of porcine serum ferric transferrin (Tf) and its stability against denaturation were studied by circular dichroism. Tf was estimated to have 19-24% alpha-helix and 50-55% beta-sheet based on the methods of Chang et al. (Chang, C.T., Wu, C.-S.C., & Yang, J.T., 1978, Anal. Biochem. 91, 13-31) and Provencher and Glöckner (Provencher, S.W. & Glöckner, J., 1981, Biochemistry 20, 33-37). Removal of the bound ferric ions (apo-Tf) did not alter the overall conformation, but there were subtle changes in local conformation based on its near-UV CD spectrum. The Tfs were stable between pH 3.5 and 11. Denaturation by guanidine hydrochloride (Gu-HCl) showed two transitions at 1.6 and 3.4 M denaturant. The process of denaturation by acid and base was reversible, whereas that by Gu-HCl was partially reversible. The irreversible thermal unfolding of Tfs began at temperatures above 60 degrees C and was not complete even at 80 degrees C. The bound irons (based on absorbance at 460 nm) were completely released at pH < 4 or in Gu-HCl solution above 1.7 M, when the protein began to unfold, but they remained intact in neutral solution even at 85 degrees C. The NH2- and COOH-terminal halves of the Tf molecule obtained by limited trypsin digestion had CD spectra similar to the spectrum of native Tf, and the COOH-terminal fragment had more stable secondary structure than the NH2-terminal fragment.     

Conserved interaction between transferrin and transferrin-binding proteins from porcine pathogens

Gram-negative porcine pathogens from the Pasteurellaceae family possess a surface receptor complex capable of acquiring iron from porcine transferrin (pTf). This receptor consists of transferrin-binding protein A (TbpA), a transmembrane iron transporter, and TbpB, a surface-exposed lipoprotein. Questions remain as to how the receptor complex engages pTf in such a way that iron is positioned for release, and whether divergent strains present distinct recognition sites on Tf. In this study, the TbpB-pTf interface was mapped using a combination of mass shift analysis and molecular docking simulations, localizing binding uniquely to the pTf C lobe for multiple divergent strains of Actinobacillus plueropneumoniae and suis. The interface was further characterized and validated with site-directed mutagenesis. Although targeting a common lobe, variants differ in preference for the two sublobes comprising the iron coordination site. Sublobes C1 and C2 participate in high affinity binding, but sublobe C1 contributes in a minor fashion to the overall affinity. Further, the TbpB-pTf complex does not release iron independent of other mediators, based on competitive iron binding studies. Together, our findings support a model whereby TbpB efficiently captures and presents iron-loaded pTf to other elements of the uptake pathway, even under low iron conditions.     

Haemophilus somnus possesses two systems for acquisition of transferrin-bound iron

Haemophilus somnus strain 649 was found to acquire iron from ovine, bovine, and goat transferrins (Tfs). Expression of Tf receptors, as evaluated by solid-phase binding assays, required the organisms to be grown under iron-restricted conditions in the presence of Tf. Competition binding assays revealed the presence of two distinct Tf-binding receptor systems, one specific for bovine Tf and the other capable of binding all three ruminant Tfs. Affinity isolation procedures using total membranes yielded three putative bovine Tf-binding polypeptides and one putative ovine and goat Tf-binding polypeptide. PCR amplification followed by DNA sequence analyses revealed that H. somnus strain 649 possesses genes that encode a bipartite TbpA-TbpB receptor along with a homolog of the Histophilus ovis single-component TbpA receptor. Expression of TbpB and the single-component TbpA would appear to be subject to a form of phase variation involving homopolymeric nucleotide tracts within the structural genes.     

Identification of human transferrin-binding sites within meningococcal transferrin-binding protein B.

Transferrin-binding protein B (TbpB) from Neisseria meningitidis binds human transferrin (hTf) at the surface of the bacterial cell as part of the iron uptake process. To identify hTf binding sites within the meningococcal TbpB, defined regions of the molecule were produced in Escherichia coli by a translational fusion expression system and the ability of the recombinant proteins (rTbpB) to bind peroxidase-conjugated hTf was characterized by Western blot and dot blot assays. Both the N-terminal domain (amino acids [aa] 2 to 351) and the C-terminal domain (aa 352 to 691) were able to bind hTf, and by a peptide spot synthesis approach, two and five hTf binding sites were identified in the N- and C-terminal domains, respectively. The hTf binding activity of three rTbpB deletion variants constructed within the central region (aa 346 to 543) highlighted the importance of a specific peptide (aa 377 to 394) in the ligand interaction. Taken together, the results indicated that the N- and C-terminal domains bound hTf approximately 10 and 1000 times less, respectively, than the full-length rTbpB (aa 2 to 691), while the central region (aa 346 to 543) had a binding avidity in the same order of magnitude as the C-terminal domain. In contrast with the hTf binding in the N-terminal domain, which was mediated by conformational epitopes, linear determinants seemed to be involved in the hTf binding in the C-terminal domain. The host specificity for transferrin appeared to be mediated by the N-terminal domain of the meningococcal rTbpB rather than the C-terminal domain, since we report that murine Tf binds to the C-terminal domain. Antisera raised to both N- and C-terminal domains were bactericidal for the parent strain, indicating that both domains are accessible at the bacterial surface. We have thus identified hTf binding sites within each domain of the TbpB from N. meningitidis and propose that the N- and C-terminal domains together contribute to the efficient binding of TbpB to hTf with their respective affinities and specificities for determinants of their ligand.     

Binding patterns of vanadium ions with different valence states to human serum transferrin studied by HPLC/high-resolution ICP-MS

Vanadium (V) is an essential metal for mammals and has different valence states. In blood, V is bound to serum transferrin (Tf), a glycoprotein which has two metal-binding sites, and carbonate is generally required for the binding. In this study, the binding patterns of V(III), V(IV), and V(V) to human serum Tf (hTf) were analyzed using an HPLC system equipped with an anion-exchange column and directly connected to a high-resolution inductively coupled plasma-mass spectrometer for metal detection (51V). In affinity to hTf, the three ions were ranked V(III)>V(IV)>V(V) in the presence of bicarbonate and V(III) reverse congruent V(IV)>V(V) in the absence. Intermediates in the "open forms" binding to the respective sites were detected at the initial stage. V(IV) and V(V) were bound to the N-lobe site in the "closed form" and "open form," respectively. In the absence of bicarbonate, V ions with respective valence states were bound to hTf in the "open form." In terms of binding to hTf, tri-valent V was most favorable in the presence of bicarbonate.     

Identification of sequences in human transferrin that bind to the bacterial receptor protein, transferrin-binding protein B

Alignment of amino-acid sequences from the N-terminal and C-terminal halves of transferrin-binding protein B revealed an underlying bilobed nature with several regions of identity. Based on this analysis, purified recombinant fusion proteins of maltose-binding protein (Mbp) with intact TbpB, its N-terminal half or C-terminal half from the human pathogens Neisseria meningitidis and Moraxella catarrhalis were produced. Solid-phase binding assays and affinity isolation assays demonstrated that the N-terminal and C-terminal halves of TbpB could bind independently to human transferrin (hTf). A solid-phase overlapping synthetic peptide library representing the amino-acid sequence of hTf was probed with soluble, labelled Mbp-TbpB fusions to localize TbpB-binding regions on hTf. An essentially identical series of peptides from domains within both lobes of hTf was recognized by intact TbpB from both organisms, demonstrating a conserved TbpB-hTf interaction. Both halves of TbpB from N. meningitidis bound the same series of peptides, which included peptides from equivalent regions on the two hTf lobes, indicating that TbpB interacts with each lobe of hTf in a similar manner. Mapping of the peptide-binding regions on a molecular model of hTf revealed a series of nearly adjacent surface regions that nearly encircled each lobe. Binding studies with chimeric hTf/bTf transferrins demonstrated that regions in the C-lobe of hTf were preferentially recognized by the N-terminal half of TbpB. Collectively, these results provide evidence that TbpB consists of two lobes, each with distinct yet homologous Tf-binding regions.     

Polymorphic variation in the amount of sialic acid attached to bovine transferrin

In this paper family studies are presented which support the hypothesis of polymorphism in the process controlling sialic acid binding to bovine transferrin which modifies its phenotype as seen in starch gel electrophoresis. It has been shown that this polymorphism is controlled by a locus Tfs with two alleles Tfs A and Tfs a. Tfs a/a animals have the abnormal phenotype with the two faster bands of the four bands of a normal transferrin allele being virtually absent. Tfs A/a and Tfs A/A are phenotypically normal. Limited evidence is presented which suggests that the Tf and Tfs loci are not linked.     

Expression of functional human transferrin in stably transfected Drosophila S2 cells

Human transferrin (hTf) is a serum glycoprotein involved in Fe3+ transport. Here, a plasmid encoding the hTf gene fused with a hexahistidine (His6) epitope tag under Drosophila metallothionein promoter (pMT) was stably transfected into Drosophila melanogaster S2 cells as a nonlytic plasmid-based system. Following 3 days of copper sulfate induction, transfected S2 cells were found to secrete hTf into serum-free culture medium at a competitively high expression level of 40.8 microg/mL, producing 6.8 microg/mL/day in a 150-mL spinner flask culture. Purification of secreted recombinant hTf using immobilized metal affinity chromatography (IMAC) yielded 95.5% pure recombinant hTf with a recovery of 32%. According to MALDI-TOF mass spectrometry analysis, purified S2 cell-derived His6-tagged recombinant hTf had a molecular weight (76.4 kDa) smaller than that of native apo-hTf (78.0 kDa). 2-Dimensional gel electrophoresis patterns showed recombinant hTf had a simpler and less acidic profile compared to that of native hTf. These data suggest recombinant hTf was incompletely (noncomplex) glycosylated and lacked sialic acids on N-glycans. However, this difference in N-glycan structure compared to native hTf had no effect on the iron-binding activity of recombinant hTf. The present data show that a plasmid-based stable transfection S2 cell system can be successfully employed as an alternative for producing secreted functional recombinant hTf.     

The stress-induced Tfs1p requires NatB-mediated acetylation to inhibit carboxypeptidase Y and to regulate the protein kinase A pathway

The Saccharomyces cerevisiae N-terminal acetyltransferase NatB consists of the subunits Nat3p and Mdm20p. We found by two-dimensional PAGE analysis that nat3Delta exhibited protein expression during growth in basal medium resembling protein expression in salt-adapted wild-type cells. The stress-induced carboxypeptidase Y (CPY) inhibitor and phosphatidylethanolamine-binding protein family member Tfs1p was identified as a novel NatB substrate. The N-terminal acetylation status of Tfs1p, Act1p, and Rnr4p in both wild type and nat3Delta was confirmed by tandem mass spectrometry. Furthermore it was found that unacetylated Tfs1p expressed in nat3Delta showed an approximately 100-fold decrease in CPY inhibition compared with the acetylated form, indicating that the N-terminal acetyl group is essential for CPY inhibition by Tfs1p. Phosphatidylethanolamine-binding proteins in other organisms have been reported to be involved in the regulation of cell signaling. Here we report that a number of proteins, whose expression has been shown previously to be dependent on the activity in the protein kinase A (PKA) signaling pathway, was found to be regulated in line with low PKA activity in the nat3Delta strain. The involvement of Nat3p and Tfs1p in PKA signaling was supported by caffeine growth inhibition studies. First, growth inhibition by caffeine addition (resulting in enhanced cAMP levels) was suppressed in tfs1Delta. Second, this suppression by tfs1Delta was abolished in the nat3Delta background, indicating that Tfs1p was not functional in the nat3Delta strain possibly because of a lack of N-terminal acetylation. We conclude that the NatB-dependent acetylation of Tfs1p appears to be essential for its inhibitory activity on CPY as well its role in regulating the PKA pathway.     

Differed preferential iron-binding lobe in human transferrin depending on the presence of bicarbonate detected by HPLC/high-resolution inductively coupled plasma mass spectrometry

The binding of iron (Fe) to human serum transferrin (Tf) was analyzed with an HPLC system equipped with an anion exchange column and directly connected with a high-resolution inductively coupled plasma mass spectrometer for metal detection. The (56)Fe level in the eluate was monitored at resolution m/Deltam=3000. Two monoferric Tfs were assigned based on the results of urea-PAGE and desferrioxamine experiments. When Fe was added as Fe-citrate stepwise to an apo-Tf solution in the presence of bicarbonate, the N-lobe site was the preferential Fe-binding site, while the C-lobe site was preferred in the absence of bicarbonate. In both cases, the Fe-peak areas of the preferential site and Fe(2)-Tf increased up to an Fe/Tf molar ratio of 1, and then the peak area of the monoferric Tf decreased while the peak area of Fe(2)-Tf increased. When the Fe/Tf molar ratio was below 1, the amount of Fe bound to the lobe with a weaker affinity was higher in Fe(2)-Tf than in the monoferric Tf in each case. Namely, Fe(2)-Tf was the preferential binding state of Fe to human serum Tf. The preference is reasonable for transferring Fe ions effectively to Tf-receptors.