The pH dependent properties of metallotransferrins: a comparative study

The dependence on pH of the absorption and circular dichroic spectra of iron(III), cobalt(III) and copper(II) transferrins has been (re)investigated. In the alkaline region, the CD profiles of iron(III) and cobalt(III) transferrin are essentially pH independent up to pH 11; only for very high pH values (pH > 11) is breakdown of the cobalt(III) and iron(III) transferrin derivatives observed, without evidence of conformational rearrangements. By contrast, the CD profiles of copper transferrin show drastic changes in shape around pH 10; these spectral changes, which are fitted to a pK_a of ~10.4, are interpreted in terms of a substantial rearrangement of the local environment of the copper ions at high pH. Although the CD spectra of copper transferrin at alkaline pH strictly resemble those observed upon addition of modifier anions, the mechanism of site destabilization in the two cases is different; at variance with the case of modifier anions, our results suggest that the high pH form of copper transferrin still contains the synergistic anion. A¹³C NMR experiment has confirmed this view. In the acidic region, iron(III) and cobalt(III) transferrins are stable down to pH ~6. For lower pH values progressive metal detachment is observed without evidence of conformational changes; around pH 4.5 most bound metals are released. In the case of the less stable copper-transferrin, metal removal from the specific binding sites is already complete around pH 6.0; in concomitance with release from the primary sites, binding of copper ions to secondary sites is observed. Additional information has been gained from CD experiments in the far UV. The pH dependent properties of iron(III), cobalt(III) and copper(II) transferrin are discussed in the frame of the present knowledge of transferrin chemistry, particular emphasis being attributed to the comparison between tripositive and bipositive metal derivatives.     

Comparison of the binding behavior of several histidine-containing proteins with immobilized copper(II) complexes of 1,4,7-triazacyclononane and 1,4-bis(1,4,7-triazacyclononan-1-yl)butane

The protein binding characteristics of the immobilized binucleating chelate system, 1,4-bis(1,4,7-triazacyclononan-1-yl)butane (tacn(2)butane), complexed with Cu(2+) ions have been investigated with hen egg white lysozyme, horse skeletal muscle myoglobin and horse heart cytochrome C, as well as three histidine-rich proteins, serum albumin, transferrin, and α(2)-macroglobulin, present in partially fractionated human serum. The effects of pH, ionic strength and elution buffers on protein binding have been examined and compared with those of the analogous immobilized mononuclear copper complex of 1,4,7-triazacyclononane (tacn). The Cu(2+)-tacn(2)butane system was generally found to exhibit higher protein binding affinities than the Cu(2+)-tacn system, suggesting that the presence of immobilized binuclear copper(II) species leads to enhanced coordinative interaction with surface-exposed amino acid residues of the studied proteins. However, under some buffer conditions the dependencies of protein binding and elution on pH and ionic strength with these immobilized metal ion affinity chromatographic (IMAC) systems were consistent with electrostatic, hydrophobic and π-bonding interactions playing a significant secondary role in addition to the dominant coordinative interactions. As such, the results indicated that the selectivities were not solely dependent on the histidine content of the protein. In accord with this conclusion, differences in the selectivities of the Cu(2+)-tacn and Cu(2+)-tacn(2)butane adsorbents for serum albumin, transferrin, and α(2)-macroglobulin were observed depending on the choice of elution buffer. This attribute suggests that additional selectivity features can be realised for the separation of specific proteins with this new class of adsorbent.     

Receptor-induced switch in site-site cooperativity during iron release by transferrin.

Iron removal by PPi from the N- and C-terminal binding sites of both free and receptor-complexed transferrin, when the partner site remains occupied with kinetically inert Co(III), has been studied at pH 7.4 and 5.6, at 25 degrees C. At extracellular pH, 7.4, the C-terminal site of free mixed-metal proteins is slightly more labile than its N-terminal counterpart in releasing iron to 0.05 M PPi. The rate and extent of iron removal are retarded from both sites when transferrins are receptor-bound. At endosomal pH, 5.6, the two sites exhibit greater kinetic heterogeneity in iron release to 0.005 M PPi. The N-terminal site is 6 times more facile in relinquishing iron than the C-terminal site when mixed-metal transferrins are free. However, the two sites are affected oppositely upon binding to the receptor. Iron release from the C-terminal site of receptor-complexed CoN-transferrin-FeC is 4 times faster than that from receptor-free protein. In contrast, iron removal from the N-terminal site of receptor-complexed FeN-transferrin-CoC is slowed by a factor of 2 compared to that from free protein. These results help explain our previous observation of a receptor-induced switch in site lability during iron removal from diferric transferrin at pH 5.6 (Bali & Aisen, 1991). Site-site cooperative interactions between the two sites of doubly-occupied transferrin during iron release are altered upon binding to receptor at pH 5.6. Iron in the otherwise weaker binding site of the N-terminal lobe is stabilized, while iron in the relatively stable binding site of the C-terminal lobe is labilized.     

Structural and functional stability of the mature transferrin receptor from human placenta

The transferrin receptor (TfR) is a N- and O-glycosylated transmembrane protein mediating the cellular iron uptake by binding and internalization of diferric transferrin. In this study, rate constants and dissociation constants of 125I-ferri-transferrin binding to the human TfR were examined dependent on receptor glycan composition, pH, bivalent cations, and temperature. To do so, purified human placental TfR was noncovalently immobilized to polystyrene surfaces and subjected to alterations in various parameters. We found that transferrin binding was clearly dependent on a receptor pretreatment with buffers of various pH in that most of the TfR molecules irreversibly lost transferrin binding activity below pH 6.5. However, the dissociation constant of the remaining active binding sites was not affected. Similarly, we were able to define the thermal stability of the receptor as a function of transferrin binding ability. Binding of transferrin was completely lost provided that the receptor was pretreated at temperatures of at least 65 degrees C. Treatment with EDTA also caused an irreversible loss of transferrin binding activity, indicating that the functionally active conformation of the mature TfR depends on bivalent cations. In order to examine the role of the receptor glycans, we enzymatically removed the sialic acid residues, the hybrid and oligomannosidic N-glycans, or all types of N-glycans. In contrast to the parameters described above, all desialylated and N-deglycosylated TfR variants had exactly the same transferrin binding properties as the native TfR. To assess changes in the secondary structure of the receptor, circular dichroic spectra were recorded from TfR at pH 5.0, from heat pretreated receptor and from deglycosylated TfR. Since the receptor did not exhibit detectable changes in the CD spectrum of the deglycosylated receptor, it can be concluded that the N-linked carbohydrates of the mature, fully processed TfR are not essential for transferrin binding and conformational stability.     

Electron spin resonance and magnetic relaxation studies of gadolinium(III) complexes with human transferrin

A human serum transferrin complex was prepared in which Gd(III) was substituted for Fe(III) at the two metal-binding sites. Characteristic changes upon metal binding in both the UV absorption of ligated tyrosines and the solvent proton longitudinal magnetic relaxation rates demonstrated 2/1 metal stoichiometry and pH-dependent binding constants. Binding studies were complicated both by binding of Gd(III) to nonspecific sites on transferrin at pH less than or equal to 7 and by complexation of the Gd(III) by the requisite bicarbonate anion at pH greater than or equal to 6.0. A unique Gd(III) electron spin resonance spectrum, with a prominent signal at g = 4.96, was observed for the specific Gd(III)-transferrin complex. The major features of this spectrum were fit successfully by a model Hamiltonian which utilized crystal field parameters similar to those determined for Fe(III) in transferrin [Aasa, R. (1970) J. Chem. Phys. 52, 3919-3924]. The magnetic field dependence of the solvent proton relaxation rate was measured as a function of both pH and metal ion concentration. An observed biphasic dependence of the relaxation rate on metal concentration is attributed to either sequential metal binding to the two iron-binding sites with different relaxation properties or random binding to two sites that are similar but show conformationally induced changes in relaxation properties as the second metal is bound. The increase in the solvent proton relaxation rate with pH is consistent with a model in which a proton of a second coordination sphere water molecule is hydrogen bonded to a metal ligand which becomes deprotonated at pH 8.5.     

High-affinity binding by the periplasmic iron-binding protein from Haemophilus influenzae is required for acquiring iron from transferrin

The periplasmic iron-binding protein, FbpA (ferric-ion-binding protein A), performs an essential role in iron acquisition from transferrin in Haemophilus influenzae. A series of site-directed mutants in the metal-binding amino acids of FbpA were prepared to determine their relative contribution to iron binding and transport. Structural studies demonstrated that the mutant proteins crystallized in an open conformation with the iron atom associated with the C-terminal domain. The iron-binding properties of the mutant proteins were assessed by several assays, including a novel competitive iron-binding assay. The relative ability of the proteins to compete for iron was pH dependent, with a rank order at pH 6.5 of wild-type, Q58L, H9Q>H9A, E57A>Y195A, Y196A. The genes encoding the mutant FbpA were introduced into H. influenzae and the resulting strains varied in the level of ferric citrate required to support growth on iron-limited medium, suggesting a rank order for metal-binding affinities under physiological conditions comparable with the competitive binding assay at pH 6.5 (wild-type=Q58L>H9Q>H9A, E57A>Y195A, Y196A). Growth dependence on human transferrin was only obtained with cells expressing wild-type, Q58L or H9Q FbpAs, proteins with stability constants derived from the competition assay >2.0x10(18) M(-1). These results suggest that a relatively high affinity of iron binding by FbpA is required for removal of iron from transferrin and its transport across the outer membrane.     

Ferrous iron release from transferrin by human neutrophil-derived superoxide anion: effect of pH and iron saturation.

The ability of superoxide anion (O2-) from stimulated human neutrophils (PMNs) to release ferrous iron (Fe2+) from transferrin was assessed. At pH 7.4, unstimulated PMNs released minimal amounts of O2- and failed to facilitate the release of Fe2+ from holosaturated transferrin. In contrast, incubation of phorbol myristate acetate (PMA)-stimulated PMNs with holosaturated transferrin at pH 7.4 enhanced the release of Fe2+ from transferrin eightfold in association with marked generation of O2-. The release of Fe2+ was inhibited by addition of superoxide dismutase (SOD), indicating that the release of Fe2+ was dependent on PMN-derived extracellular O2-. In contrast, at physiologic pH (7.4), incubation of transferrin at physiological levels of iron saturation (e.g. 32%) with unstimulated or PMA stimulated PMNs failed to facilitate the release of Fe2+. The effect of decreasing the pH on the release of Fe2+ from transferrin by PMN-derived O2- was determined. Decreasing the pH greatly facilitated the release of Fe2+ from both holosaturated transferrin and from transferrin at physiological levels of iron saturation by PMN-derived O2-. Release of Fe2+ occurred despite a decrease in the amount of extracellular O2- generated by PMNs in an acidic environment. These results suggest that transferrin at physiologic levels of iron saturation may serve as a source of Fe2+ for biological reactions in disease states where activated phagocytes are present and there is a decrease in tissue pH. The unbound iron could participate in biological reactions including promoting propagation of lipid peroxidation reactions or hydroxyl radical formation following reaction with phagocytic cell-derived hydrogen peroxide.     

Amonabactin-mediated iron acquisition from transferrin and lactoferrin by <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> : direct measurement of individual microscopic rate constants

 The effectiveness and mechanism of iron acquisition from transferrin or lactoferrin by Aeromonas hydrophila has been analyzed with regard to the pathogenesis of this microbe. The ability of A. hydrophila's siderophore, amonabactin, to remove iron from transferrin was evaluated with in vitro competition experiments. The kinetics of iron removal from the three molecular forms of ferric transferrin (diferric, N- and C-terminal monoferric) were investigated by separating each form by urea gel electrophoresis. The first direct determination of individual microscopic rates of iron removal from diferric transferrin is a result. A. hydrophila 495A2 was cultured in an iron-starved defined medium and the growth monitored. Addition of transferrin or lactoferrin promoted bacterial growth. Growth promotion was independent of the level of transferrin or lactoferrin iron saturation (between 30 and 100%), even when the protein was sequestered inside dialysis tubing. Siderophore production was also increased when transferrin or lactoferrin was enclosed in a dialysis tube. Cell yield and growth rate were identical in experiments where transferrin was present inside or outside the dialysis tube, indicating that binding of transferrin was not essential and that the siderophore plays a major role in iron uptake from transferrin. The rate of iron removal from diferric transferrin shows a hyperbolic dependence on amonabactin concentration. Surprisingly, amonabactin cannot remove iron from the more weakly binding N-terminal site of monoferric transferrin, while it is able to remove iron from the more strongly binding C-terminal site of monoferric transferrin. Iron from both sites is removed from diferric transferrin and it is the N-terminal site (which does not release iron in the monoferric protein) that releases iron more rapidly! It is apparent that there is a significant interaction of the two lobes of the protein with regard to the chelator access. Taken together, these results support an amonabactin-dependent mechanism for iron removal by A. hydrophila from transferrin and lactoferrin. The implications of these findings for an amonabactin-dependent mechanism for iron removal by A. hydrophila from transferrin and lactoferrin are discussed. Received: 8 August 1999 / Accepted: 22 October 1999     

A new role for the transferrin receptor in the release of iron from transferrin

Iron removal by pyrophosphate from human serum diferric transferrin and the complex of transferrin with its receptor was studied in 0.05 M HEPES or MES buffers containing 0.1 M NaCl and 0.01 M CHAPS at 25 degrees C at pH 7.4, 6.4, and 5.6. At each pH, the concentration of pyrophosphate was adjusted to achieve rates of release amenable to study over a reasonable time course. Released iron was separated from protein-bound iron by poly(ethylene glycol) precipitation of aliquots drawn from the reaction mixture at various times during the course of a kinetic run. The amount of 59Fe label associated with the protein and pyrophosphate was determined from the radioactivity of precipitate and supernatant, respectively, in each aliquot. Iron removal of 0.05 M pyrophosphate at pH 7.4 from diferric transferrin bound to the receptor is considerably slower than that from free diferric transferrin, with observed pseudo-first-order rate constants of 0.020 and 0.191 min-1, respectively. For iron removal by 0.01 M pyrophosphate at pH 6.4, corresponding rate constants are 0.031 and 0.644 min-1. However, at pH 5.6, iron removal by 0.001 M pyrophosphate is faster from diferric transferrin bound to its receptor than from free transferrin (observed rate constants of 0.819 and 0.160 min-1, respectively). Thus, the transferrin receptor not only facilitates the removal of iron from diferric transferrin at the low pH that prevails in endocytic vesicles but may also reduce its accessibility to iron acceptors at extracellular pH, thereby minimizing the likelihood of nonspecific release of iron from transferrin at the cell surface.     

Effect of pH and iron content of transferrin on its binding to reticulocyte receptors

The effect of pH on the binding of apotransferrin and diferric transferrin to reticulocyte membrane receptors was investigated using rabbit transferrin and rabbit reticulocyte ghosts, intact cells and a detergent-solubilized extract of reticulocyte membranes. The studies were performed within the pH range 4.5–8.0. The binding of apotransferrin to ghosts and membrane extracts and its uptake by intact reticulocytes was high at pH levels below 6.5 but decreased to very low values as the pH was raised above 6.5. By contrast, diferric transferrin showed a high level of binding and uptake between pH 7.0 and 8.0 in addition to binding only slightly less than did apotransferrin at pH values below 6.5. It is proposed that the high affinity of apotransferrin for its receptor at lower pH values and low affinity at pH 7.0 or above allow transferrin to remain bound to the receptor when it is within acidic intracellular vesicles, even after loss of its iron, but also allow ready release from the cell membrane when it is exteriorized by exocytosis after iron uptake. The binding of transferrin to the receptor throughout the endocytosis-exocytosis cycle may protect it from proteolytic breakdown and aid in its recycling to the outer cell membrane     

Bioaccumulation of metal cations by Saccharomyces cerevisiae

Yeast cells are capable of accumulation of various heavy metals, preferentially accumulating those of potential toxicity and also those of value. They retain their ability to accumulate heavy metals under a wide range of ambient conditions. In the present study it was shown that yeast cells in suspension accumulate heavy metal cations such as Cu²⁺, Co²⁺. The level of copper accumulation was dependent on the ambient metal concentration and was markedly inhibited by extremes of ambient pH. Temperature (5–40°C) and the presence of the alkali metal sodium had much smaller effects on the level of copper accumulation. This suggests that in waste-waters of pH 5.0–9.0, yeast biomass could provide an effective bioaccumlator for removal and/or recovery of the metal. During bioaccumulation and subsequent processes it is necessary to retain the biomass. It was shown in the present study that this could be achieved by cell immobilization. Immobilization allowed for complete removal of Cu²⁺, Co²⁺, and Cd²⁺ from synthetic metal solutions. The immobilized material could be freed of metals by use of the chelating agent ethylenediamine tetraacetic acid (EDTA) and recycled for further bioaccumulation events with little loss of accumulation capacity.Correspondence to: J. R. Duncan     

A QM/MM study of the complexes formed by aluminum and iron with serum transferrin at neutral and acidic pH

Serum transferrin (sTf) transports iron in serum and internalizes in cells via receptor mediated endocytosis. Additionally, sTf has been identified as the predominant aluminum carrier in serum. Some questions remain unclear about the exact mechanism for the metal release or whether the aluminum and iron show the same binding mode during the entire process. In the present work, simulation techniques at quantum and atomic levels have been employed in order to gain access into a molecular level understanding of the metal-bound sTf complex, and to describe the binding of Al(III) and Fe(III) ions to sTf. First, hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations were carried out in order to analyze the dynamics of the aluminum-loaded complex, taking into account the different pH conditions in blood and into the cell. Moreover, the complexes formed by transferrin with Al(III) and Fe(III) were optimized with high level density functional theory (DFT)/MM methods. All these results indicate that the interaction mode of Al(III) and Fe(III) with sTf change upon different pH conditions, and that the coordination of Al(III) and Fe(III) is not equivalent during the metal intake, transport and release processes. Our results emphasize the importance of the pH on the metal binding and release mechanism and suggest that Al(III) can follow the iron pathway to get access into cells, although once there, it may show a different binding mode, leading to a different mechanism for its release.     

The influence of uncoordinated histidines on iron release from transferrin. A chemical modification study

Histidine residues that influence the chelate-mediated removal of iron from transferrin have been investigated. Diferric human serum transferrin was chemically modified to various extents using ethoxyformic anhydride, a reagent for histidines. A kinetic analysis of the modification reaction revealed the presence of a fast reacting pool of 9 +/- .8 histidine residues and a slow reacting pool of 5.8 +/- .6 residues. There are 18 histidine residues in transferrin. The rates of modification of the two pools differed by a factor of 5. The pyrophosphate-mediated removal of iron from the two binding sites of native and partially modified transferrins was studied at pH 6.9 using desferrioximine B as a terminal iron acceptor. Under these conditions, the rate of iron removal from the NH2-terminal site was about six times faster than from the COOH-terminal site. Both rates were significantly reduced, i.e. by a factor of approximately 6-8, upon complete ethoxyformylation of all reactive histidines on the protein. The kinetic data of partially modified transferrins were analyzed by the Tsou Chen-Lu statistical method; the results are consistent with the hypothesis that modification of a single uncoordinated histidine in each of the two iron binding domains stabilizes the protein kinetically against loss of iron. The dependence of the iron removal reaction on pH is consistent with such an interpretation. The putative histidines, although not ligands, may be close to the metal in both binding sites, thus influencing the rate of iron removal by pyrophosphate. These histidines belong to the pool of rapidly modified residues and thus are readily accessible to solvent and chelators.     

Receptor-modulated iron release from transferrin: differential effects on N- and C-terminal sites

Iron release to PPi from N- and C-terminal monoferric transferrins and their complexes with transferrin receptor has been studied at pH 7.4 and 5.6 in 0.05 M HEPES or MES/0.1 M NaCl/0.01 M CHAPS at 25 degrees C. The two sites exhibit kinetic heterogeneity in releasing iron. The N-terminal form is slightly less labile than its C-terminal counterpart at pH 7.4, but much more facile in releasing iron at pH 5.6. At pH 7.4, iron removal by 0.05 M pyrophosphate from each form of monoferric transferrin complexed to the receptor is considerably slower than from the corresponding free monoferric transferrin. However, at pH 5.6, complexation of transferrin to its receptor affects the two forms differently. The rate of iron release to 0.005 M pyrophosphate by the N-terminal species is substantially the same whether transferrin is free or bound to the receptor. In contrast, the C-terminal form releases iron much faster when complexed to the receptor than when free. Urea/PAGE analysis of iron removal from free and receptor-complexed diferric transferrin at pH 5.6 reveals that its C-terminal site is also more labile in the complex, but its N-terminal site is more labile in free diferric transferrin. Thus, the newly discovered role of transferrin receptor in modulating iron release from transferrin predominantly involves the C-terminal site. This observation helps explain the prevalence of circulating N-terminal monoferric transferrin in the human circulation.     

Comaprative binding study of aluminum and chromium to human transferrin

The characteristics of aluminum and chromium binding to apotransferrin (apo-tf) have been investigated and compared. Both metal ions were taken up by human transferrin forming complexes with the maximum absorbances at 405 nm for chromium-transferrin (cr-tf) and 240 nm for aluminum-transferrin (Al-tf). In the presence of citric acid, chromium binding to transferrin is five times more than aluminum. The binding of aluminum or chromium to apo-transferrin was reduced by 18 and 22% in the presence of 200 ng/mL of iron. The binding of both metals to apo-tf appears to be pH dependent. In acidic pHs, less chromium and more aluminum binding occurred.     

An in vitro study on the binding of Al(III) to human serum transferrin with the isoelectric focusing technique

Transferrin saturated with Al³⁺ subjected to isoelectric focusing (IEF) in a pH gradient can be separated into four fractions, representing the apotransferrin, transferrin with aluminum at the metal binding site in the C- or N-terminal lobe, or both. The electrophoretic mobilities of these four fractions are identical to those of the iron-transferrin counterparts. Simultaneous binding of aluminum and iron to transferrin can also be demonstrated. The decreased saturation after IEF indicates that the affinity of transferrin for aluminum is low compared with its affinity for iron. This effect is particularly evident when bicarbonate is used as the synergistic anion in the loading procedure. In contrast, loading of transferrin with aluminum in the presence of oxalate produces a di-aluminum-transferrin complex that is stable during IEF.     

A ligand-receptor binding assay by receptor immobilization

Using transferrin-transferrin receptor binding as a model of ligand-receptor binding, we have developed a new and simple binding assay for the solubilized receptor. Solubilized membrane proteins containing transferrin receptor were immobilized by covalent binding to beads having chemical reactive epoxide groups, and then 125I-labeled transferrin was added to the beads. Dose-dependent, ligand-specific, and saturable binding of 125I-labeled transferrin to the immobilized membrane proteins were demonstrated and a Scatchard analysis derived affinity of Kd = 1.8 X 10(-9) M was obtained. These results indicate that the immobilization of receptors onto beads may be useful in a simple binding assay of the solubilized receptor.     

Release of iron from transferrin by phosphonocarboxylate and diphosphonate chelating agents

The rates at which phosphonocarboxylate and diphosphonate ligands remove iron from the serum iron transport protein transferrin at 25 degrees C and pH 7.4 have been evaluated. These ligands show a combination of saturation and first-order kinetics with respect to the free ligand concentrations. The ability of the ligands to remove iron from transferrin appears to be subject to steric restrictions that are essentially identical to those associated with the ability of a ligand to substitute for the synergistic carbonate anion. This observation supports the hypothesis that the first-order component for iron removal involves a mechanism in which the rate-limiting step is the slow substitution of the synergistic carbonate by the incoming chelating agent. Studies on monoferric transferrins indicate that phosphonocarboxylates are unusually effective at removing iron from the C-terminal site of the protein. Difference UV spectroscopy has been used to show that the phosphonocarboxylates bind strongly to apotransferrin. It is suggested that the rapid release of iron from the C-terminal site may be due to the binding of the ligand to an allosteric anion-binding site in the C-terminal lobe of the protein.     

Inhibition of reticulocyte iron uptake by NH4Cl and CH3NH2

The aim of this investigation was to test the hypothesis that elevation of intracellular pH would inhibit iron uptake by reticulocytes. The experiments were performed with rabbit reticulocytes and iron bound to rabbit transferrin. Incubation of the cells with NH₄Cl, (NH₄)₂CO₃, CH₃NH₂ and (CH₃)₂NH was used in an attempt to increase intracellular pH. These substances were all found to inhibit iron uptake by reticulocytes. The mechanism of action of NH₄Cl and CH₃NH₂ was investigated in detail. Similar results were found with both reagents. They inhibited iron uptake in a concentration-dependent manner, but produced a small increase in the cellular uptake of transferrin. The onset of action was rapid and the effect was reversible. There was no decrease in the number of transferrin-binding sites per cell and their apparent affinity for transferrin increased slightly, while the efficiency of iron removal from transferrin per binding site diminished greatly. The rate of transferrin release from reticulocytes was unaffected. NH₄Cl did not affect the rate of iron release from transferrin in a cell-free system. Incubation of reticulocytes with 10 mM NH₄Cl or CH₃NH₂ was found to produce an increase in intracellular pH of 0.05—0.15 pH units. The intracellular pH determined by used of the weak acid 5,5-dimethyl-oxazolidine-2,4-dione was significantly higher than that obtained with the weak base (CH₃)₂NH. By transmission electron microscopy it was shown that reticulocytes treated with NH₄Cl or CH₃NH₂ have enlarged intracellular vesicles. The results are considered to support the hypothesis that iron release from transferrin in reticulocytes occurs as a result of protonation of the transferrin within intracellular vesicles. According to this hypothesis, weak bases such as NH₃ and CH₃NH₂ inhibit iron release by neutralizing H⁺ within the vesicles.     

Hepatic heparan sulphate proteoglycan and the recycling of transferrin.

Heparan sulphate proteoglycan, labelled with [35S]sulphate, was prepared from rat livers for studies of its interaction with purified rat transferrin. Affinity chromatography of the preparation on columns of immobilized differic transferrin and apotransferrin showed that the proteoglycan possessed affinity for both types of matrices at pH 7.3 and that this affinity significantly increased at pH 5.6. The glycosaminoglycan chains liberated from the proteoglycan by heparan sulphate lyase also bound to apotransferrin, albeit less strongly, whereas the deglycosylated core protein exhibited virtually no interaction with this matrix. In the presence of the proteoglycan at pH 5.6, the release of iron from the N-lobe of transferrin was accelerated. These observations suggest that heparan sulphate proteoglycan from the liver can mimick some of the known functions of bona fide transferrin receptors and, hence, interaction with the proteoglycan may provide an alternative nondegradative pathway for transferrin through hepatic cells.