Identification of platination sites on human serum transferrin using 13C and 15N NMR spectroscopy

 Reactions between various apo and metal-bound forms of human serum transferrin (80 kDa) and the recombinant N-lobe (40 kDa) with [Pt(en)Cl₂] or cis-[PtCl₂(NH₃)₂] have been investigated in solution via observation of [¹H,¹⁵N] NMR resonances of the Pt complexes, [¹H,¹³C] resonances of the eCH₃ groups of the protein methionine residues, and by chromatographic analysis of single-site methionine mutants. For the whole protein, the preferred Pt binding site appears to be Met256. Additional binding occurs at the other surface-exposed methionine (Met499), which is platinated at a slower rate than Met256. In contrast, binding of similar Pt compounds to the N-lobe of the protein occurs at Met313, rather than Met256. Met313 is buried in the interlobe contact region of intact transferrin. After loss of one chloride ligand from Pt and binding to methionine sulfur of the N-lobe, chelate-ring closure appears to occur with binding to a deprotonated backbone amide nitrogen, and the loss of the other chloride ligand. Such chelate-ring closure was not observed during reactions of the whole protein, even after several days. Received: 5 May 1999 / Accepted: 26 July 1999     

Binding of bismuth to serum proteins: implication for targets of Bi(III) in blood plasma

Bismuth complexes have been widely used in clinical treatment as antiulcer drugs. However, different adverse effects have been observed and the diagnosis is generally confirmed by the detection of bismuth in blood or blood plasma. In this study, binding of bismuth to human serum albumin was studied by fluorescence spectroscopy with the binding constant logK(a) to be 11.2. Competitive binding of bismuth to human albumin and transferrin was carried out at pH 7.4 by FPLC and ICP-MS. It was found that over 70% of bismuth binds to transferrin even in the presence of a large excess of albumin (albumin/transferrin=13:1) at pH 7.4, 10 mM bicarbonate. The distribution of bismuth between the two proteins was almost unchanged when Cys(34) of albumin was blocked. However, all bismuth binds to albumin when iron-saturated transferrin was used. Almost all of the bismuth was distributed over the fractions containing transferrin (70%) and albumin (<30%) in serum. The percentage of bismuth associated with transferrin was further increased by 15% with elevated transferrin in serum. Binding of bismuth to transferrin is much stronger than human albumin. Transferrin is probably the major target of bismuth in blood plasma, and it may play a role in the pharmacology of bismuth.     

Binding of manganese in human and rat plasma

Albumin, transferrin and 'transmanganin' have all been proposed as the major Mn-binding ligand in plasma. The present investigations were initiated in order to resolve these discrepancies. Compared to other metals tested (109 Cd2+, 65Zn2+, 59Fe3+), 54Mn2+ bound poorly to purified albumin. The addition of exogenous albumin to plasma did not result in an increased 54Mn radioactivity associated with this protein. Also, incubation of 65Zn-albumin in the presence of excess Mn2+ (1 mM) did not result in the displacement of Zn from albumin or Mn binding. In contrast to these results, 54Mn was bound to purified transferrin, not as readily as Fe3+, but better than Zn2+ or Cd2+. Saturation of transferrin with Fe3+ (1.6 micrograms Fe/mg) prevented the binding of 54Mn indicating that Mn probably binds to Fe-binding sites on the protein. Polyacrylamide gel electrophoresis further demonstrated the association of 54Mn with transferrin rather than with albumin in both human and rat plasma. The amount of 54Mn radioactivity recovered with transferrin increased as incubation time was increased, probably due to oxidation of Mn2+ to Mn3+. Mn binding to transferrin reached a maximum within 5 and 12 h of incubation. About 50% of 54Mn migrated with transferrin, whereas only 5% was associated with albumin. A significant portion (20-55%) of the 54Mn radioactivity migrated with electrophoretically slow plasma components whose identity was not determined. Possibilities include alpha 2-macroglobulin, heavy gamma-globulins and/or heavy lipoproteins.     

Crystal structure of the N-lobe of lactoferrin binding protein B from Moraxella bovis

Lactoferrin (Lf) is a bi-lobed, iron-binding protein found on mucosal surfaces and at sites of inflammation. Gram-negative pathogens from the Neisseriaceae and Moraxellaceae families are capable of using Lf as a source of iron for growth through a process mediated by a bacterial surface receptor that directly binds host Lf. This receptor consists of an integral outer membrane protein, lactoferrin binding protein A (LbpA), and a surface lipoprotein, lactoferrin binding protein B (LbpB). The N-lobe of the homologous transferrin binding protein B, TbpB, has been shown to facilitate transferrin binding in the process of iron acquisition. Currently there is little known about the role of LbpB in iron acquisition or how Lf interacts with the bacterial receptor proteins. No structural information on any LbpB or domain is available. In this study, we express and purify from Escherichia coli the full-length LbpB and the N-lobe of LbpB from the bovine pathogen Moraxella bovis for crystallization trials. We demonstrate that M. bovis LbpB binds to bovine but not human Lf. We also report the crystal structure of the N-terminal lobe of LbpB from M. bovis and compare it with the published structures of TbpB to speculate on the process of Lf mediated iron acquisition.     

Structure of the human transferrin receptor-transferrin complex

Iron, insoluble as free Fe(3+) and toxic as free Fe(2+), is distributed through the body as Fe(3+) bound to transferrin (Tf) for delivery to cells by endocytosis of its complex with transferrin receptor (TfR). Although much is understood of the transferrin endocytotic cycle, little has been uncovered of the molecular details underlying the formation of the receptor-transferrin complex. Using cryo-electron microscopy, we have produced a density map of the TfR-Tf complex at subnanometer resolution. An atomic model, obtained by fitting crystal structures of diferric Tf and the receptor ectodomain into the map, shows that the Tf N-lobe is sandwiched between the membrane and the TfR ectodomain and that the C-lobe abuts the receptor helical domain. When Tf binds receptor, its N-lobe moves by about 9 A with respect to its C-lobe. The structure of TfR-Tf complex helps account for known differences in the iron-release properties of free and receptor bound Tf.     

Vanadium complexes of transferrin and ferritin in the rat

Vanadium associates with serum transferrin of rats administered vanadyl(IV) sulfate or ammonium metavanadate(V) by gastric intubation. Low molecular weight species account for only 3% of the vanadium present in plasma. The element distributes between the two major isotransferrins in proportion to their concentrations. Rat apotransferrin binds both vanadium(IV) and vanadium(V), forming 2:1 metal-protein complexes in both instances. Although the two isotransferrins apparently differ in their physiological properties, they exhibit identical vanadyl(IV) (VO2+) EPR spectra, indicating identical or very similar metal binding sites for both proteins. In contrast to other transferrins, the two sites of the rat protein are spectroscopically indistinguishable and exhibit a VO2+ EPR spectrum similar to that of the C-terminal metal binding site of human serum transferrin. VO2+ EPR signals are observed with liver, spleen, and kidney tissue samples from animals maintained on a vanadium-supplemented diet. These signals arise from a specific intracellular VO2+ complex with the iron storage protein ferritin.     

In calmodulin-IQ domain complexes, the Ca(2+)-free and Ca(2+)-bound forms of the calmodulin C-lobe direct the N-lobe to different binding sites

We have investigated the roles played by the calmodulin (CaM) N- and C-lobes in establishing the conformations of CaM-IQ domain complexes in different Ca(2+)-free and Ca(2+)-bound states. Our results indicate a dominant role for the C-lobe in these complexes. When the C-lobe is Ca(2+)-free, it directs the N-lobe to a binding site within the IQ domain consensus sequence. It appears that the N-lobe must be Ca(2+)-free to interact productively with this site. When the C-lobe is Ca(2+)-bound, it directs the N-lobe to a site upstream of the consensus sequence, and it appears that the N-lobe must be Ca(2+)-bound to interact productively with this site. A model for switching in CaM-IQ domain complexes is presented in which the N-lobe adopts bound and extended positions that depend on the status of the Ca(2+)-binding sites in each CaM lobe and the compositions of the two N-lobe binding sites. Ca(2+)-dependent changes in the conformation of the bound C-lobe that appear to be responsible for directed N-lobe binding are also identified. Changes in the equilibria between extended and bound N-lobe positions may control bridging interactions in which the extended N-lobe is bound to another CaM-binding domain. Ca(2+)-dependent control of bridging interactions with CaM has been implicated in the regulation of ion channel and unconventional myosin activities.     

Flurorometric study of the partition of bilirubin among blood components: basis for rapid microassays of bilirubin and bilirubin binding capacity in whole blood

Bilirubin binds to many sites in blood, the strongest binding being to a single site on albumin. Secondary sites on albumin, most sites on other plasma proteins, and sites on erythrocyte membranes have affinities for bilirubin that are at most one-hundredth as great. Bilirubin binds to hemoglobin in red cells with an effective affinity that is less than one-thousandth that of the primary albumin site. Essentially the only bilirubin present in blood which fluoresces is that bound to the primary albumin site. Almost all the other bilirubin in blood fluoresces with a yield no more than one-fiftieth as large. Quantitative fluorometry of whole blood is possible using the “front-face” technique. The concentration of bilirubin bound to the primary albumin site can be determined in this way. The albumin binding capacity of a blood specimen can be similarly assayed upon titration of the specimen with bilirubin. The nonionic detergent dodecyldimethylamine oxide (DDAO) scavenges bilirubin from all sites in blood, and, since bilirubin is fluorescent in DDAO micelles, the total blood bilirubin can be assayed fluorometrically after addition of DDAO to the specimen. This detergent method also allows facile assay of red-cell-bound bilirubin. These fluorometric assays for total blood bilirubin, albumin-bound bilirubin, and albumin binding capacity are simple and rapid and use very small volumes of blood. They should be of great value in the research on neonatal jaundice and in its clinical management.     

Mutational analysis of C-lobe ligands of human serum transferrin: insights into the mechanism of iron release

Each homologous lobe of human serum transferrin (hTF) has one Fe(3+) ion bound by an aspartic acid, a histidine, two tyrosine residues, and two oxygens from the synergistic anion, carbonate. Extensive characterization of these ligands in the N-terminal lobe has been carried out. Despite sharing the same set of ligands, there is a substantial amount of evidence that the N- and C-lobes are inequivalent. Studies of full-length hTF have shown that iron release from each lobe is kinetically distinguishable. To simplify the assessment of mutations in the C-lobe, we have created mutant hTF molecules in which the N-lobe binds iron with high affinity or not at all. Mutations targeting the C-lobe liganding residues have been introduced into these hTF constructs. UV-visible spectral, kinetic, and EPR studies have been undertaken to assess the effects of each mutation and to allow direct comparison to the N-lobe. As found for the N-lobe, the presence of Y517 in the C-lobe (equivalent to Y188 in the N-lobe) is absolutely essential for the binding of iron. Unlike the N-lobe, however, mutation of Y426 (equivalent to Y95) does not produce a stable complex with iron. For the mutants that retain the ability to bind iron (D392S and H585A), the rates of release are considerably slower than those measured for equivalent mutations in the N-lobe at both pH 7.4 and pH 5.6. Equilibrium binding experiments with HeLa S(3) cells indicate that recombinant hTF, in which Y426 or H585 is mutated, favor a closed or nearly closed conformation while those with mutations of the D392 or Y517 ligands appear to promote an open conformation. The differences in the effects of mutating the liganding residues in the two lobes and the subtle indications of cooperativity between lobes point to the importance of the transferrin receptor in effecting iron release from the C-lobe. Significantly, the equilibrium binding experiments also indicate that, regardless of which lobe contains the iron, the free energy of binding is equivalent and not additive; each monoferric hTF has a free energy of binding that is 82% of diferric hTF.     

Titanium preferential binding sites in human serum transferrin at physiological concentrations

Serum transferrin (Tf) is an iron binding glycoprotein that plays a central role in the metabolism of this essential metal but it also binds other metal ions. Four main transferrin forms containing different iron binding states can be distinguished in human serum samples: monoferric (C-site or N-site), holotransferrin (with two Fe atoms) and apotransferrin (with no metal). Recently, it has been reported that Tf binds also Ti even more tightly than does Fe, in artificially Ti(iv) spiked solutions. However, very limited work has been done on the Ti binding to Tf at physiological concentrations in patients carrying intramedullary Ti nails. Here we report the chemical association of Ti to Tf "in vivo" under different chromatographic conditions by elemental mass spectrometry using double focusing inductively coupled plasma (DF-ICP-MS) as detector. For the separation of the Ti/Fe-Tf forms different gradient conditions have been explored. The observed results reveal that human serum Ti (from patients carrying intramedullary Ti nails) is uniquely associated to the N-lobe of Tf. The investigation of the influence of sialic acid in the carbohydrate chain of human serum Tf, studied by incubating the protein with neuraminidase (sialidase) to obtain the monosialilated species, revealed that the binding affinity of Ti was similar for monosialo-Tf and for native-Tf and occurs in the N-lobe. These results suggest that the species Fe(C)Ti(N)-TF might provide a route for Ti entry into cells via the transferrin receptors after the release of the metal from its implants.     

Unusual features for zirconium(IV) binding to human serum transferrin

Human serum apotransferrin (hTF) binds to Zr(IV) slowly in the presence of nitrilotriacetate (NTA), citrate or ethylenediaminetetraacetate (EDTA) as donor ligands. For Zr(NTA)(2)(2-) as donor, equilibrium was reached in ca. 2 h (pH 7.4, 298 K, 10 mM Hepes, 5 mM bicarbonate) and full loading of the N- and C-lobe sites was achievable to give Zr(2)-hTF. (13)C NMR data suggest that carbonate can bind as a synergistic anion. (1)H and 2D [(1)H,(13)C] (using epsilon-[(13)C]Met-hTF) NMR studies show that there is little lobe-selectively in the order of Zr(IV) uptake. Fe(III) displaced Zr(IV) from the C-lobe of Zr(2)-hTF first, followed by the N-lobe. However, in the presence of a large excess of NTA, Zr(IV) binds to the N-lobe of holo-hTF (Fe(2)-hTF) first followed by the C-lobe. The (1)H and (13)C NMR chemical shift changes for epsilon-[(13)CH(3)] of Met464, which is close to the C-lobe site, are quite distinct from those observed previously for Al(III), Fe(III), Ti(IV), Ga(III) and Bi(III) binding to hTF, suggesting that Zr(IV) binding may not induce lobe closure [as observed previously for Hf(IV)]. This may affect receptor recognition and play a role in the different biological behaviour of Zr(IV) compared to Ti(IV).     

Evolution of the transferrin family: conservation of residues associated with iron and anion binding

The transferrin family spans both vertebrates and invertebrates. It includes serum transferrin, ovotransferrin, lactoferrin, melanotransferrin, inhibitor of carbonic anhydrase, saxiphilin, the major yolk protein in sea urchins, the crayfish protein, pacifastin, and a protein from green algae. Most (but not all) contain two domains of around 340 residues, thought to have evolved from an ancient duplication event. For serum transferrin, ovotransferrin and lactoferrin each of the duplicated lobes binds one atom of Fe (III) and one carbonate anion. With a few notable exceptions each iron atom is coordinated to four conserved amino acid residues: an aspartic acid, two tyrosines, and a histidine, while anion binding is associated with an arginine and a threonine in close proximity. These six residues in each lobe were examined for their evolutionary conservation in the homologous N- and C-lobes of 82 complete transferrin sequences from 61 different species. Of the ligands in the N-lobe, the histidine ligand shows the most variability in sequence. Also, of note, four of the twelve insect transferrins have glutamic acid substituted for aspartic acid in the N-lobe (as seen in the bacterial ferric binding proteins). In addition, there is a wide spread substitution of lysine for the anion binding arginine in the N-lobe in many organisms including all of the fish, the sea squirt and many of the unusual family members i.e., saxiphilin and the green alga protein. It is hoped that this short analysis will provide the impetus to establish the true function of some of the TF family members that clearly lack the ability to bind iron in one or both lobes and additionally clarify the evolutionary history of this important family of proteins.     

Cadmium-binding to transferrin in the plasma of the common carp Cyprinus carpio

In this study, it was investigated by autoradiography with radioactive cadmium after Western blotting of two-dimensional electrophoresis gels, to which proteins cadmium is mainly bound in plasma of common carp Cyprinus carpio. The obtained results demonstrate that in carp plasma, cadmium is primarily bound to two high molecular weight proteins. Relative small amounts are bound to a protein with M(r) approximately 60000. The other metal-binding protein, with M(r) approximately 70000 and pI approximately 6.7 was identified as transferrin. The conditional equilibrium constants for the binding of cadmium ions to the two metal-binding sites of this protein were calculated as logK(1)=5.40+/-0.12 and logK(2)=4.66+/-0.21, which are comparable to those of human transferrin under the same experimental conditions. Transport of cadmium in plasma of carp was found to be different from that of brown trout Salmo trutta and man, where cadmium is mainly bound to albumin and transferrin. The prominent binding of cadmium to transferrin can be explained by the absence or at least the very low concentrations in which albumin is present in carp plasma.     

Peptide-peptide interactions between human transferrin and transferrin-binding protein B from Moraxella catarrhalis

Transferrin-binding protein B (TbpB) is one component of a bipartite receptor in several gram-negative bacterial species that binds host transferrin and mediates the uptake of iron for growth. Transferrin and TbpB are both bilobed proteins, and the interaction between these proteins seems to involve similar lobe-lobe interactions. Synthetic overlapping peptide libraries representing the N lobe of TbpB from Moraxella catarrhalis were prepared and probed with labeled human transferrin. Transferrin-binding peptides were localized to six different regions of the TbpB N lobe, and reciprocal experiments identified six different regions of the C lobe of transferrin that bound TbpB. Truncations of the N lobe of TbpB that sequentially removed each transferrin-binding determinant were used to probe an overlapping peptide library of the C lobe of human transferrin. The removal of each TbpB N-lobe transferrin-binding determinant resulted in a loss of reactivity with peptides from the synthetic peptide library representing the C lobe of transferrin. Thus, individual peptide-peptide interactions between ligand and receptor were identified. A structural model of human transferrin was used to map surface regions capable of binding to TbpB.     

Complexation of ytterbium to human transferrin and its uptake by K562 cells.

There is an increasing interest in the use of lanthanides in medicine. However, the mechanism of their accumulation in cells is not well understood. Lanthanide cations are similar to ferric ions with regard to transferrin binding, suggesting transferrin-receptor mediated transport is possible; however, this has not yet been confirmed. In order to clarify this mechanism, we investigated the binding of Yb3+ to apotransferrin by UV-Vis spectroscopy and stopped-flow spectrophotometry, and found that Yb3+ binds to apotransferrin at the specific iron sites in the presence of bicarbonate. The apparent binding constants of these sites showed that the affinity of Yb3+ is lower than that of Fe3+and binding of Yb3+ in the N-lobe is kinetically favored while the C-lobe is thermodynamically favored. The first Yb3+ bound to the C-lobe quantitatively with a Yb/apotransferrin molar ratio of < 1, whereas the binding to the other site is weaker and approaches completeness by a higher molar ratio only. As demonstrated by 1H NMR spectra, Yb3+ binding disturbed the conformation of apotransferrin in a manner similar to Fe3+. Flow cytometric studies on the uptake of fluorescein isothiocyanate labeled Yb3+-bound transferrin species by K562 cells showed that they bind to the cell receptors. Laser scanning confocal microscopic studies with fluorescein isothiocyanate labeled Yb3+-bound transferrin and propidium iodide labeled DNA and RNA in cells indicated that the Yb3+ entered the cells. The Yb3+-transferrin complex inhibited the uptake of the fluorescein labeled ferric-saturated transferrin (Fe2-transferrin) complex into K562 cells. The results demonstrate that the complex of Yb3+-transferrin complex was recognized by the transferrin receptor and that the transferrin-receptor-mediated mechanism is a possible pathway for Yb3+ accumulation in cells.     

Structural and functional consequences of binding site mutations in transferrin: crystal structures of the Asp63Glu and Arg124Ala mutants of the N-lobe of human transferrin

Human transferrin is a serum protein whose function is to bind Fe(3+) with very high affinity and transport it to cells, for delivery by receptor-mediated endocytosis. Structurally, the transferrin molecule is folded into two globular lobes, representing its N-terminal and C-terminal halves, with each lobe possessing a high-affinity iron binding site, in a cleft between two domains. Central to function is a highly conserved set of iron ligands, including an aspartate residue (Asp63 in the N-lobe) that also hydrogen bonds between the two domains and an arginine residue (Arg124 in the N-lobe) that binds an iron-bound carbonate ion. To further probe the roles of these residues, we have determined the crystal structures of the D63E and R124A mutants of the N-terminal half-molecule of human transferrin. The structure of the D63E mutant, determined at 1.9 A resolution (R = 0.245, R(free) = 0.261), showed that the carboxyl group still binds to iron despite the larger size of the Glu side chain, with some slight rearrangement of the first turn of alpha-helix residues 63-72, to which it is attached. The structure of the R124A mutant, determined at 2.4 A resolution (R = 0.219, R(free) = 0.288), shows that the loss of the arginine side chain results in a 0.3 A displacement of the carbonate ion, and an accompanying movement of the iron atom. In both mutants, the iron coordination is changed slightly, the principal change being in each case a lengthening of the Fe-N(His249) bond. Both mutants also release iron more readily than the wild type, kinetically and in terms of acid lability of iron binding. We attribute this to more facile protonation of the synergistically bound carbonate ion, in the case of R124A, and to strain resulting from the accommodation of the larger Glu side chain, in the case of D63E. In both cases, the weakened Fe-N(His) bond may also contribute, consistent with protonation of the His ligand being an early intermediate step in iron release, following the protonation of the carbonate ion.     

Apocalmodulin and Ca2+ calmodulin-binding sites on the CaV1.2 channel

The cardiac L-type voltage-dependent calcium channel is responsible for initiating excitation-contraction coupling. Three sequences (amino acids 1609-1628, 1627-1652, and 1665-1685, designated A, C, and IQ, respectively) of its alpha(1) subunit contribute to calmodulin (CaM) binding and Ca(2+)-dependent inactivation. Peptides matching the A, C, and IQ sequences all bind Ca(2+)CaM. Longer peptides representing A plus C (A-C) or C plus IQ (C-IQ) bind only a single molecule of Ca(2+)CaM. Apocalmodulin (ApoCaM) binds with low affinity to the IQ peptide and with higher affinity to the C-IQ peptide. Binding to the IQ and C peptides increases the Ca(2+) affinity of the C-lobe of CaM, but only the IQ peptide alters the Ca(2+) affinity of the N-lobe. Conversion of the isoleucine and glutamine residues of the IQ motif to alanines in the channel destroys inactivation (Zühlke et al., 2000). The double mutation in the peptide reduces the interaction with apoCaM. A mutant CaM unable to bind Ca(2+) at sites 3 and 4 (which abolishes the ability of CaM to inactivate the channel) binds to the IQ, but not to the C or A peptide. Our data are consistent with a model in which apoCaM binding to the region around the IQ motif is necessary for the rapid binding of Ca(2+) to the C-lobe of CaM. Upon Ca(2+) binding, this lobe is likely to engage the A-C region.     

Comparison of binding interactions of lomefloxacin to serum albumin and serum transferrin by resonance light scattering and fluorescence quenching methods

The interaction between lomefloxacin (LMF) and two drug carrier proteins, human serum albumin (HSA) and serum transferrin (TF), were studied and compared by fluorescence quenching, resonance light scattering (RLS), and circular dichroism (CD) spectroscopic along with molecular modeling. Fluorescence data show that LMF has a stronger quenching effect on HSA than on TF. The binding constant and the number of binding sites were calculated as 6.00 x 10(5) M(-1) and 0.77 for HSA, and 4.66 x 10(5) M(-1) and 1.02, for TF, respectively. Also, these binding parameters were calculated by RLS data, as a novel approach and were compared to that obtained from fluorescence. The micro-environment changes of Trp residues were evident in both proteins. The quantitative analysis of the secondary structure in both proteins further confirmed the drug-induced conformational changes. The distance (r) between donors (HSA and TF) and acceptor (LMF) were obtained by fluorescence resonance energy transfer (FRET) theory and found to be 1.83 nm and 1.71 nm for HSA and TF respectively. Moreover, molecular modeling studies suggested the sub-domain IB in HSA and N-lobe in TF as the candidate place for the formation of the binding site of LMF on these proteins.     

Protein binding and stability of norepinephrine in human blood plasma. Involvement of prealbumin, alpha 1-acid glycoprotein and albumin

The binding of norepinephrine (NE) to plasma proteins of fresh human blood obtained from healthy volunteers was studied by ultrafiltration at different NE concentrations and incubation times at 37 degrees C. At 1.7 nM L-[3H]-NE binding was approximately 25%. The binding was rapid and was not influenced by the incubation time. [3H]-NE could be dissociated from its binding sites by acid precipitation and, after HPLC, showed to be unchanged NE. No difference in NE binding was found between plasma collected in EGTA-GSH or heparin solution. There was no degradation of NE when incubated in plasma at 37 degrees C for 10 h, even without the addition of antioxidants. Therefore, in the present study, binding represented interaction of unchanged NE with plasma proteins. The whole plasma binding was saturable over the range of 0.66 nM to 0.59 mM of NE. Scatchard plot of specific binding revealed high-affinity sites with a Kd of 5.4 nM and a Bmax of 3.9 fmoles.mg-1 protein, and low-affinity sites with a Kd of 2.7 microM and a Bmax of 3.3 pmoles.mg-1 protein. Electrophoretic characterization of NE-binding proteins showed that about 60% of bound NE was associated to albumin, and 20% to prealbumin. NE binding to pure human plasma proteins was also studied using ultrafiltration. Scatchard analyses revealed a single class of very high-affinity binding sites for prealbumin (Kd 4.9 nM), a single class of binding sites for alpha 1-acid glycoprotein (Kd 54 microM) and two classes of binding sites for albumin with high (Kd 1.7 microM) and low (Kd 0.8 mM) affinities respectively. The main results obtained in this study - a) reversibility of NE binding, b) stability of free and bound NE in plasma, c) involvement of the prealbumin as a specific binding protein - point out to a specific transport for NE in human blood plasma.     

A kinetic study on the distribution of Cu(II)-ions between albumin and transferrin

Serum albumin (human, bovine) has a specific Cu(II)-ion binding site, and is proposed to act as a copper transport protein in blood plasma. Human transferrin, normally about 30% saturated with iron in vivo, has two sites/molecule capable of complexing Cu(II); one more strongly than the other (Hirose et al. 1996). The present study shows that this binding site has a slightly stronger affinity for Cu(II) than that on the albumins. However, both human- and bovine albumin could take up part of the transferrin bound Cu(II), the second order rate constant for the reaction estimated to 12 mM(-1) min(-1) for both species. In vivo the albumin concentration is considerably higher than that of iron-free transferrin, and it seems unlikely that the latter can compete with albumin for non-ceruloplasmin cupric ions.