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Ferritins are highly conserved supramolecular protein nanostructures that play a key role in iron homeostasis. Thousands of iron atoms can be stored inside their hollow cavity as a hydrated ferric oxyhydroxide mineral. Although phosphate associates with the ferritin iron nanoparticles, the effect of physiological concentrations on the kinetics, structure, and reactivity of ferritin iron cores has not yet been explored. Here, the iron loading and mobilization kinetics were studied in the presence of 1-10 mM phosphate using homopolymer and heteropolymer ferritins having different H to L subunit ratios. In the absence of ferritin, phosphate enhances the rate of ferrous ion oxidation and forms large and soluble polymeric Fe(III)-phosphate species. In the presence of phosphate, Fe(II) oxidation and core formation in ferritin is significantly accelerated with oxidation rates several-fold higher than with phosphate alone. High-angle annular dark-field scanning transmission electron microscopy measurements revealed a strong phosphate effect on both the size and morphology of the iron mineral in H-rich (but not L-rich) ferritins. While iron nanoparticles in L-rich ferritins have spherical shape in the absence and presence of phosphate, iron nanoparticles in H-rich ferritins change from irregular shapes in the absence of phosphate to spherical particles in the presence of phosphate with larger size distribution and smaller particle size. In the presence of phosphate, the kinetics of iron-reductive mobilization from ferritin releases twice as much iron than in its absence. Altogether, our results demonstrate an important role for phosphate, and the ferritin H and L subunit composition toward the kinetics of iron oxidation and removal from ferritin, as well as the structure and reactivity of the iron mineral, and may have an important implication on ferritin iron management

Mammalian ferritins are predominantly heteropolymeric species consisting of 2 structurally similar, but functionally and genetically distinct subunit types, called H (Heavy) and L (Light). The two subunits co-assemble in different H and L ratios to form 24-mer shell-like protein nanocages where thousands of iron atoms can be mineralized inside a hollow cavity. Here, we use differential scanning calorimetry (DSC) to study ferritin stability and understand how various combinations of H and L subunits confer aspects of protein structure-function relationships. Using a recently engineered plasmid design that enables the synthesis of complex ferritin nanostructures with specific H to L subunit ratios, we show that homopolymer L and heteropolymer L-rich ferritins have a remarkable hyperthermostability (Tm = 115 ± 1°C) compared to their H-ferritin homologues (Tm = 93 ± 1°C). Our data reveal a significant linear correlation between protein thermal stability and the number of L subunits present on the ferritin shell. A strong and unexpected iron-induced protein thermal destabilization effect (ΔT

The physical properties of in vitro iron-reconstituted and genetically engineered human heteropolymer ferritins were investigated.

Ferritinophagy is a ferritin autophagic degradation process mediated by the selective nuclear receptor coactivator-4 (NCOA4). NCOA4 binds to ferritin and delivers it to nascent autophagosomes, which then merge with the lysosomes for ferritin degradation and iron release. Earlier studies have demonstrated a specific association of NCOA4 with ferritin H-subunits, but not L-subunits. However, neither the thermodynamics of this interaction nor the effect of NCOA4 on iron oxidation, iron mineral core formation, or iron mobilization in ferritin has been explored. Using isothermal titration calorimetry, light absorption spectroscopy, and a soluble fragment (residues 383-522) of human NCOA4 expressed in Escherichia coli, we show that the NCOA4 fragment specifically binds H-rich ferritins with a binding stoichiometry of ∼8 NCOA4 molecules per ferritin shell, and Kd values of ∼0.4 and ∼2 μM for homopolymer H-chain ferritin and heteropolymer H-rich ferritin, respectively. The binding reaction was both enthalpically and entropically favored. Whereas the iron oxidation kinetics were not affected by the presence of NCOA4, iron mobilization from ferritin by two different reducing agents (FMN/NADH and sodium dithionite) showed a strong inhibitory effect that was dependent on the concentration of NCOA4 present in solution. Our results suggest that the binding of NCOA4 to ferritin may interfere in the electron transfer pathway through the ferritin shell and may have important biological implications on cellular iron homeostasis.

Most in vitro iron mobilization studies from ferritin have been performed in aqueous buffered solutions using a variety of reducing substances. The kinetics of iron mobilization from ferritin in a medium that resembles the complex milieu of cells could dramatically differ from those in aqueous solutions, and to our knowledge, no such studies have been performed. Here, we have studied the kinetics of iron release from ferritin in fresh yeast cell lysates and examined the effect of cellular metabolites on this process. Our results show that iron release from ferritin in buffer is extremely slow compared to cell lysate under identical experimental conditions, suggesting that certain cellular metabolites present in yeast cell lysate facilitate the reductive release of ferric iron from the ferritin core. Using filtration membranes with different molecular weight cut-offs (3, 10, 30, 50, and 100 kDa), we demonstrate that a cellular component >50 kDa is implicated in the reductive release of iron. When the cell lysate was washed three times with buffer, or when NADPH was omitted from the solution, a dramatic decrease in iron mobilization rates was observed. The addition of physiological concentrations of free flavins, such as FMN, FAD, and riboflavin showed about a two-fold increase in the amount of released iron. Notably, all iron release kinetics occurred while the solution oxygen level was still high. Altogether, our results indicate that in addition to ferritin proteolysis, there exists an auxiliary iron reductive mechanism that involves long-range electron transfer reactions facilitated by the ferritin shell. The physiological implications of such iron reductive mechanisms are discussed.

The development of safe and effective vaccines against SARS-CoV-2 and other viruses with high antigenic drift is of crucial importance to public health. Ferritin is a well characterized and ubiquitous iron storage protein that has emerged not only as a useful nanoreactor and nanocarrier, but more recently as an efficient platform for vaccine development.This review discusses ferritin structure-function properties, self-assembly, and novel bioengineering strategies such as interior cavity and exterior surface modifications for cargo encapsulation and delivery. It also discusses the use of ferritin as a scaffold for biomedical applications, especially for vaccine development against influenza, Epstein-Barr, HIV, hepatitis-C, Lyme disease, and respiratory viruses such as SARS-CoV-2. The use of ferritin for the synthesis of mosaic vaccines to deliver a cocktail of antigens that elicit broad immune protection against different viral variants is also explored.The remarkable stability, biocompatibility, surface functionalization, and self-assembly properties of ferritin nanoparticles make them very attractive platforms for a wide range of biomedical applications, including the development of vaccines. Strong immune responses have been observed in pre-clinical studies against a wide range of pathogens and have led to the exploration of ferritin nanoparticles-based vaccines in multiple phase I clinical trials.The broad protective antibody response of ferritin nanoparticles-based vaccines demonstrates the usefulness of ferritin as a highly promising and effective approaches for vaccine development.

Mammalian ferritins are predominantly heteropolymeric species consisting of 24 structurally similar, but functionally different subunit types, named H and L, that co-assemble in different proportions. Despite their discovery more than 8 decades ago, recombinant human heteropolymer ferritins have never been synthesized, owing to the lack of a good expression system. Here, we describe for the first time a unique approach that uses a novel plasmid design that enables the synthesis of these complex ferritin nanostructures. Our study reveals an original system that can be easily tuned by altering the concentrations of two inducers, allowing the synthesis of a full spectrum of heteropolymer ferritins, from H-rich to L-rich ferritins and any combinations in-between (isoferritins). The H to L subunit composition of purified ferritin heteropolymers was analyzed by SDS-PAGE and capillary gel electrophoresis, and their iron handling properties characterized by light absorption spectroscopy. Our novel approach allows future investigations of the structural and functional differences of isoferritin populations, which remain largely obscure. This is particularly exciting since a change in the ferritin H- to L-subunit ratio could potentially lead to new iron core morphologies for various applications in bio-nanotechnologies.

Mushrooms are a potent source of dietary antioxidants and are often marketed for their capability of decreasing oxidative stress and preventing diseases. To date, however, little research has been done on the antioxidant activity of commercially available mushroom extractions, or their stability over time. Herein, the antioxidant capacity of six commercially available mushroom tinctures (hydro-alcoholic extracts of chaga, maitake, shiitake, reishi, lion's mane, and turkey tail) were evaluated using a panel of five antioxidant assays: Oxygen Radical Absorbance Capacity (ORAC), Nanoceria Reducing Antioxidant Capacity (NanoCerac), DPPH radical scavenging, Total Phenolic Content (TPC), and Ferric Reducing Antioxidant Power (FRAP). Commercial samples were compared to our own laboratory preparations of the same species, in a preliminary assessment of extraction technique and antioxidant stability. Mushroom extracts exhibited different antioxidant capacities depending on species and extraction method (e.g., 0.023 to 0.63 μmol TE/mg for commercial ORAC vs. 0.05 to 1.8 μmol TE/mg for laboratory ORAC for all species). In all cases, laboratory extracts exhibited considerably higher antioxidant activities than commercial extracts. In both extract types, chaga and maitake consistently demonstrated higher antioxidant activity than the other mushrooms. Preliminary antioxidant stability testing of our samples revealed stable ORAC values for one month at room temperature, followed by a 48% decrease after 4 months. Our results provide important insights regarding the effects of extraction techniques and storage conditions, on the antioxidant activity in mushroom extracts, particularly those made and sold commercially.

Fenretinide is a synthetic retinoid pharmaceutical linked to ceramide build-up in vivo. Saposin D is an intralysosomal protein necessary for ceramide binding/degradation. We show, via electronic absorption spectroscopy, fluorescence spectroscopy, and ceramide hydrolysis assays, that fenretinide is bound by saposin D {K(a) = (1.45 ± 0.49) × 10(5) M(–1)}, and affects ceramide solubilization/degradation.

Background Mag-Fluo-4 is increasingly employed for studying Ca2+ signaling in skeletal muscle; however, the lack of information on the Ca2+-Mag-Fluo-4 reaction limits its wider usage. Methods Fluorescence and isothermal titration calorimetry (ITC) experiments were performed to determine the binding stoichiometry (n) and thermodynamics (enthalpy (ΔH) and entropy (ΔS) changes), as well as the in vitro and in situ Kd of the Ca2+-Mag-Fluo-4 reaction. Rate constants (kon, koff), fluorescence maximum (Fmax), minimum (Fmin), and the dye compartmentalization were also estimated. Experiments in cells used enzymatically dissociated flexor digitorum brevis fibres of C57BL6, adult mice, loaded at room temperature for 8 min, with 6 μM Mag-Fluo-4, AM, and permeabilized with saponin or ionomycin. All measurements were done at 20 °C. Results The in vitro fluorescence assays showed a binding stoichiometry of 0.5 for the Ca2+/Mag-Fluo-4 (n = 5) reaction. ITC results (n = 3) provided ΔH and ΔS values of 2.3 (0.7) kJ/mol and 97.8 (5.9) J/mol.K, respectively. The in situ Kd was 1.652 × 105μM2(n = 58 fibres, R2 = 0.99). With an Fmax of 150.9 (8.8) A.U. (n = 8), Fmin of 0.14 (0.1) A.U. (n = 10), and ΔF of Ca2+ transients of 8.4 (2.5) A.U. (n = 10), the sarcoplasmic [Ca2+]peak reached 22.5 (7.8) μM. Compartmentalized dye amounted to only 1.1 (0.7)% (n = 10). CONCLUSIONS: Two Mag-Fluo-4 molecules coalesce around one Ca2+ ion, in an entropy-driven, very low in situ affinity reaction, making it suitable to reliably track the kinetics of rapid muscle Ca2+ transients. General significance Our results may be relevant to the quantitative study of Ca2+ kinetics in many other cell types.

Background Ferritins are ubiquitous multi-subunit iron storage and detoxification proteins that play a critical role in iron homeostasis. Ferrous ions that enter the protein's shell through hydrophilic channels are rapidly oxidized at dinuclear centers on the H-subunit before transfer to the protein's cavity for storage. The mechanisms of iron loading have been extensively studied, but little is known about iron mobilization. Fe(III) reduction can occur via rapid reduction by suitable reducing agents followed by chelation of Fe(II) ions or via direct and slow Fe(III) chelation. Here, the iron release kinetics from ferritin by FMNH2 in the presence of various chaotropic agents are studied and their in-vivo physiological significance discussed. Methods The iron release kinetics from horse and human ferritins by FMNH2 were monitored at 522 nm where the Fe(II)–bipyridine complex absorbs. The experiments were performed in the presence of different concentrations of three chaotropic agents, urea, guanidine HCl, and triton. Results and conclusions Under our experimental conditions, iron reductive mobilization by the non-enzymatic FMN/NAD(P)H system is limited by the concentration of FMNH2 and is independent on the type or amount of chaotropes present. Diffusion of FMNH2 through the ferritin pores is an unlikely mechanism for ferritin iron reduction. An iron mobilization mechanism involving rapid electron transfer through the protein shell is discussed. General significance Caution must be exercised when interpreting the kinetics of iron mobilization from ferritin using the FMN/NAD(P)H system. The kinetics are highly dependent on the amount of dissolved oxygen and the concentration of reagents used.

In mammals, the iron storage and detoxification protein ferritin is composed of two functionally and genetically distinct subunit types, H (heavy) and L (light). The two subunits co-assemble in various ratios, with a tissue specific distribution, to form shell-like protein structures of 24 subunits within which a mineralized iron core is stored. The H-subunits possess ferroxidase centers that catalyze the rapid oxidation of ferrous ions, whereas the L-subunit does not have such centers and is believed to play an important role in electron transfer reactions that occur during the uptake and release of iron. Pathogenic mutations on the L-chain lead to neuroferritinopathy, a neurodegenerative disease characterized by abnormal accumulation of ferritin inclusion bodies and iron in the central nervous system. Here, we have characterized the thermal stability, iron loading capacity, iron uptake, and iron release properties of ferritin heteropolymers carrying the three pathogenic L-ferritin mutants (L154fs, L167fs, and L148fs, which for simplicity we named Ln1, Ln2 and Ln3, respectively), and a non-pathogenic variant (L135P) bearing a single substitution on the 3-fold axes of L-subunits. The UV-Vis data show a similar iron loading capacity (ranging between 1800 to 2400 Fe(iii)/shell) for all ferritin samples examined in this study, with Ln2 holding the least amount of iron (i.e. 1800 Fe(iii)/shell). The three pathogenic L-ferritin mutants revealed higher rates of iron oxidation and iron release, suggesting that a few mutated L-chains on the heteropolymer have a significant effect on iron permeability through the ferritin shell. DSC thermograms showed a strong destabilization effect, the severity of which depends on the location of the frameshift mutations (i.e. wt heteropolymer ferritin ≅ homopolymer H-chain > L135P > Ln2 > Ln1 > Ln3). Variant L135P had only minor effects on the protein functionality and stability, suggesting that local melting of the 3-fold axes in this variant may not be responsible for neuroferritinopathy-like disorders. The data support the hypothesis that hereditary neuroferritinopathies are due to alterations of ferritin functionality and lower physical stability which correlate with the frameshifts introduced at the C-terminal sequence and explain the dominant transmission of the disorder.

The excessively high and inconsistent literature values for Km,Fe and Km,O2 prompted us to examine the iron oxidation kinetics in ferritin, the major iron storage protein in mammals, and to determine whether a traditional Michaelis–Menten enzymatic behavior is obeyed. The kinetics of Fe(ii) oxidation and mineralization catalyzed by three different types of ferritins (recombinant human homopolymer 24H, HuHF, human heteropolymer ∼21H:3L, HL, and horse spleen heteropolymer ∼3.3H:20.7L, HosF) were therefore studied under physiologically relevant O2 concentrations, but also in the presence of excess Fe(ii) and O2 concentrations. The observed iron oxidation kinetics exhibited two distinct phases (phase I and phase II), neither of which obeyed Michaelis–Menten kinetics. While phase I was very rapid and corresponded to the oxidation of approximately 2 Fe(ii) ions per H-subunit, phase II was much slower and varied linearly with the concentration of iron(ii) cations in solution, independent of the size of the iron core. Under low oxygen concentration close to physiological, the iron uptake kinetics revealed a Michaelis–Menten behavior with Km,O2 values in the low μM range (i.e. ∼1–2 μM range). Our experimental Km,O2 values are significantly lower than typical cellular oxygen concentration, indicating that iron oxidation and mineralization in ferritin should not be affected by the oxygenation level of cells, and should proceed even under hypoxic events. A kinetic model is proposed in which the inhibition of the protein’s activity is caused by bound iron(iii) cations at the ferroxidase center, with the rate limiting step corresponding to an exchange or a displacement reaction between incoming Fe(ii) cations and bound Fe(iii) cations.

Iron is an essential nutrient for virtually all forms of life. Because of its redox properties and involvement in a wide range of biological processes, a number of qualitative and quantitative chemical tools have been developed to detect reduced (Fe2+) and oxidized (Fe3+) forms of iron in biomolecules. These types of measurements are not only important in detecting iron species in solution, but also in understanding iron distribution, accumulation, and role in physiological and pathological processes. Here, we use UV–vis spectrophotometry and three common chromogenic reagents, ferrozine, 2,2′-bipyridine, and 1,10-phenanthroline to detect and quantify the concentration of ferrous ions in aqueous solutions, owing to the unique absorption spectra, specific molar absorptivity, and characteristic colors of these Fe2+-chelator complexes. Our results show that the kinetics of the formation of the {Fe2+-(ferrozine)3} complex, but not the{Fe2+-(bipyridine)3} or the {Fe(II)-(phenanthroline)3} complexes depend on the concentration of the iron chelator, requiring up to 20 min to complete when close to stoichiometric ratios are employed. The molar absorptivity values of these complexes under excess chelator concentrations were ~ 10% to 15% higher than reported literature values (i.e. 31,500 ± 1500 M−1 cm−1 for ferrozine at 562 nm, 9950 ± 100 M−1 cm−1 for 2,2′-bipyridine at 522 nm, and 12,450 ± 370 M−1 cm−1 for 1,10-phenanthroline at 510 nm). Our results have important implications when quantifying iron in biological systems and reveal optimal experimental conditions that must be employed for the accurate measurements of ferrous ions, whether free in solution, or after reduction of protein-bound ferric ions.

Capillary zone electrophoresis (CZE) is a sensitive and rapid technique for determining traces of inorganic cations in water samples. CZE with indirect UV-diode array detection (CZE-DAD) was utilized to identify several inorganic cations in natural, potable, and wastewater samples. A pH 4.35 background electrolyte system was employed and consisted of 15 mM imidazole, 8 mM malonic acid, 2 mM 18-crown-6 ether as complexing agents, 10% v/v methanol as an organic modifier with indirect absorbance reference at 214 nm. The CZE method involved electromigration injection at 5 kV for 5 s, a separation voltage of 20 kV at 25°C, and a detection wavelength of 280 nm. Six main cations (ammonium NH4+, potassium K+, calcium Ca2+, sodium Na+, magnesium Mg2+, and lead Pb2+) were tested, and all but lead, were detected in the water samples at concentrations between 0.03 and 755 ppm with a detection limit ranging between 0.023 and 0.084 ppm. The successful evaluation of the proposed methodology allowed us to reliably detect and separate six metal ions in different water samples without any pretreatment. All water samples were collected from Northern New York towns and the Raquette River water system, the third longest river in New York State and the largest watershed of the central and western Adirondacks.

Serum albumins are ubiquitous proteins able to bind a variety of exogenous and endogenous ligands including hydrophobic pharmaceuticals. Most drugs bind to two very active binding regions located within sub-domains IIA and IIIA of the protein, also known as Sudlow’s sites. The drug binding mode of serum albumin provides important pharmacological information and influences drug solubility, efficacy, biological distribution, and excretion. Here, the binding thermodynamics of Diclofenac and Naproxen, two non-steroidal anti-inflammatory drugs (NSAIDs) to bovine and human serum albumins (BSA and HSA, respectively) were studied by isothermal titration calorimetry (ITC), fluorescence spectroscopy and differential scanning calorimetry (DSC). The ITC data show that the binding affinity (K) of Diclofenac to BSA and HSA is on the order of 104 M−1 with a binding stoichiometry (n) of 2 drug molecules per protein. Naproxen binding to the two proteins exhibits a different profile with K and n values on the order of 106 M−1 and 0.75 for BSA, and 105 M−1 and 3 for HSA, respectively. The binding of the two drugs to HSA is found to be both enthalpically and entropically favored suggesting the formation of hydrogen bonds and van der Waals hydrophobic effects. Binding of the two drugs to BSA is only enthalpically favored with an unfavorable entropy term. Significant enthalpy–entropy compensation phenomena were reported for Diclofenac and Naproxen binding to BSA but not to HSA. Fluorescence quenching data between Diclofenac and the two proteins suggest static collisions and the formation of ground-state protein-drug complexes. The DSC data corroborate the ITC findings and show multiple sequential unfolding events and a strong drug stabilization effect at high drug to protein ratios. Overall, the calorimetric and spectroscopic data provided insights into the nature of these protein-drugs interactions and might offer useful information in future drug discovery studies.

The thermodynamics of Cu 2+ - and Ni 2+ -NTA binding to polypeptides containing 2–6 histidine residues in neutral buffered aqueous solutions were characterized by isothermal titration calorimetry (ITC). The apparent dissociation constants of ternary complexes of N-acetylhexahistidine-Ni 2+ -NTA and N-acetylhexahistidine-Cu 2+ -NTA were 69.5 μM and 27.1 μM, respectively. Decreased number of histidine residues in the oligohistidine fragment caused gradual decrease in the stability of the mixed metal complexes and a change in the binding stoichiometry from ∼1:1 for N-acetylhexahistidine and N-acetylpentahistidine to ∼0.5:1 for N-acetyltetrahistidine and ∼0.3:1 for N-acetyltrihistidine, N-acetyldihistidine and N-acetylhistidine ligands. In all cases, the binding interactions are shown to be enthalpically favored. Other metal cations formed less stable mixed metal complexes with stability constants in the order of Cu 2+ > Ni 2+ > Mg 2+ , Co 2+ , Zn 2+ >> Mn 2+ , Ca 2+ . The significance of the data is discussed in terms of important practical applications.

Capillary zone electrophoresis (CZE) is a sensitive and rapid technique used for determining traces of inorganic and organic anions in potable, natural, and wastewaters. Here, CZE with indirect UV-diode array detection (CZE-DAD) was employed with a background electrolyte system comprising of an Agilent Technologies proprietary basic anion buffer at pH 12.0 and a forensic anion detection method. The limits of detection (LOD) for this method ranged between 3 and 5 ppm and involved hydrodynamic injection of 50 mbar for 6 s with a negative polarity separation voltage of −30 kV at 30°C, a detection wavelength of 350 nm and indirect reference of 275 nm. Fourteen different anions were checked for in the water samples that were examined and included bromide, chloride, thiosulfate, nitrate, nitrite, sulfate, azide, carbonate, fluoride, arsenate, phosphate, acetate, lactate, and silicate. The water samples were collected from Northern New York towns and the Raquette River water system, the third longest river in New York State and the largest watershed of the central and western Adirondacks. The concentrations detected for these anions ranged from

International audience; Saposin B (SapB) is a human lysosomal protein, critical for the degradation of O-sulfogalactosylceramide (sulfatide). SapB binds sulfatide and presents it to the active site of the enzyme arylsulfatase A. Deficiency of SapB leads to fatal activator-deficient metachromatic leukodystrophy. Given the conformational flexibility and the large hydrophobic "pocket" produced upon (physiologically relevant) homodimerization, SapB may have broader substrate diversity than originally thought. Herein, we present evidence using fluorescence spectroscopy and computational docking studies that SapB binds a wide variety of ligands at KD values varying from micromolar to nanomolar, with entropy being the primary driving force. We further demonstrate, for the first time, that SapB has two binding sites that can sequentially (and in a preferred order) accommodate up to two ligands at once.

Human serum transferrin (hTf) is a single-chain bilobal glycoprotein that efficiently delivers iron to mammalian cells by endocytosis via the transferrin/transferrin receptor system. While extensive studies have been directed towards the study of ferric ion binding to hTf, ferrous ion interactions with the protein have never been firmly investigated owing to the rapid oxidation of Fe(II) to Fe(III) and the difficulty in maintaining a fully anaerobic environment. Here, the binding of Fe(2+) and Zn(2+) ions to hTf has been studied under anaerobic and aerobic conditions, respectively, in the presence and absence of bicarbonate by means of isothermal titration calorimetry (ITC) and fluorescence spectroscopy. The ITC data indicate the presence of one class of strong binding sites with dissociation constants of 25.2 nM for Fe(2+) and 6.7 nM for Zn(2+) and maximum binding stoichiometries of 1 Zn(2+) (or 1 Fe(2+)) per hTf molecule. With either metal, the binding interaction was achieved by both favorable enthalpy and entropy changes (ΔH(0)~-12 kJ/mol and ΔS(0)~106 J/mol·K for Fe(2+) and ΔH(0)~-18 kJ/mol and ΔS(0)~97 J/mol·K for Zn(2+)). The large and positive entropy values are most likely due to the change in the hydration of the protein and the metal ions upon interaction. Rapid kinetics stopped-flow fluorescence spectroscopy revealed two different complexation mechanisms with different degrees of conformational changes upon metal ion binding. Our results are discussed in terms of a plausible scenario for iron dissociation from transferrin by which the highly stable Fe(3+)-hTf complex might be reduced to the more labile Fe(2+) ion before iron is released to the cytosol.

At least three ferritins are found in the bacterium Escherichia coli: the heme-containing bacterioferritin (EcBFR) and two nonheme bacterial ferritins (EcFtnA and EcFtnB). In addition to the conserved A and B sites of the diiron ferroxidase center, EcFtnA has a third iron-binding site (the C site) of unknown function that is nearby the diiron site. In the present work, the complex chemistry of iron oxidation and deposition in EcFtnA was further defined through a combination of oximetry, pH stat, stopped-flow and conventional kinetics, UV–vis, fluorescence, and EPR spectroscopic measurements on both the wild-type protein and site-directed variants of the A, B, and C sites. The data reveal that although H2O2 is a product of dioxygen reduction in EcFtnA and oxidation occurs with a stoichiometry of Fe2+/O2 ∼ 3:1 most of the H2O2 produced is consumed in subsequent reactions with a 2:1 Fe2+/H2O2 stoichiometry, thus suppressing hydroxyl-radical formation. Although the A and B sites are essential for rapid iron o...

Atovaquone is a front-line antimalarial drug that functions by competitively inhibiting binding of coenzyme Q10 to the cytochrome bc1 complex. Atovaquone is administered orally, but has low solubility and is poorly absorbed with high variability in bioavailability. In vivo binding of human serum albumin has been cited as the major transporter of atovaquone in plasma. The research presented herein demonstrates that saposin B, a known binder/transporter of coenzyme Q10, also binds to atovaquone in a 1 : 1 ratio and with comparably high affinity at pH 5.5.

In animals, the iron storage and detoxification protein, ferritin, is composed of two functionally and genetically distinct subunit types, H (heavy) and L (light), which co-assemble in various ratios with tissue specific distributions to form shell-like protein structures of 24 subunits within which a mineralized iron core is stored. The H-subunit possesses a ferroxidase center (FC) that catalyzes Fe(II) oxidation, whereas the L-subunit does not. To assess the role of the L-subunit in iron oxidation and core formation, two human recombinant heteropolymeric ferritins, designated H-rich and L-rich with ratios of ∼20H:4L and ∼22L:2H, respectively, were employed and compared to the human homopolymeric H-subunit ferritin (HuHF). These heteropolymeric ferritins have a composition similar to the composition of those found in hearts and brains (i.e., H-rich) and in livers and spleens (i.e., L-rich). As for HuHF, iron oxidation in H-rich ferritin was found to proceed with a 2:1 Fe(II):O2 stoichiometry at an iron level of 2 Fe(II) atoms/H-subunit with the generation of H2O2. The H2O2 reacted with additional Fe(II) in a 2:1 Fe(II):H2O2 ratio, thus avoiding the production of hydroxyl radical. A μ-1,2-peroxo-diFe(III) intermediate was observed at the FC of H-rich ferritin as for HuHF. Importantly, the H-rich protein regenerated full ferroxidase activity more rapidly than HuHF did and additionally formed larger iron cores, indicating dual roles for the L-subunit in facilitating iron turnover at the FC and in mineralization of the core. The L-rich ferritin, while also facilitating iron oxidation at the FC, additionally promoted oxidation at the mineral surface once the iron binding capacity of the FC was exceeded.

The thermally stable trans isomers of donor-acceptor substituted azobenzene dyes can be easily transformed into unstable cis-form by light. Although azobenzene and its derivatives have been extensively investigated over the years, many aspects of the isomerization reaction kinetics are still unclear or controversial at best. Here, we investigate the kinetics of the thermal cis-trans isomerization of 4-anilino-4'-nitroazobenzene (4A4NAB) in six solvents of different polarities by means of flash photolysis. Using the transition state theory, we were able to determine a number of thermodynamic quantities including the standard enthalpy of activation Δ‡H0, the standard entropy of activation Δ‡S0, the Gibbs free energy of activation Δ‡G0, and the quasi-equilibrium constant K‡ of the isomerization reaction. Higher rates of isomerization and lower activation energies were observed in polar media, consistent with the formation of a charged transition state complex and the presence of solute−solvent interactions such as intermolecular hydrogen bonding. The data suggest that the cis-trans thermal isomerization of 4A4NAB is highly influenced by solvent polarity and the isomerization mechanism is likely to proceed via rotation about the azo double bond.

A significant challenge associated with systemic delivery of cationic antimicrobial peptides and polymers lies in their limited hemocompatibility toward vast numbers of circulating red blood cells (RBCs). Supramolecular assembly of cationic peptides and polymers can be an effective strategy to develop an array of antimicrobial nanomaterials with tunable material structures, stability and thus optimized bioactivity to overcome some of the existing challenges associated with conventional antimicrobials. In this work, we will demonstrate the supramolecular design of self-assembling antimicrobial nanofibers (SAANs) which have tunable supramolecular nanostructures, stability, internal molecular packing and surface chemistry through self-assembly of de novo designed cationic peptides and peptide–PEG conjugates. The interaction of the SAANs with human RBCs was evaluated in a stringent biological assay (beyond a traditional hemolysis assay) where both hemolytic and eryptotic activity were examined to establish a fundamental understanding of the correlation between material structure and hemocompatibility. It was found that although the SAANs showed moderate hemolytic activities, their abilities to induce eryptosis vary significantly and are much more sensitive to the internal molecular packing, supramolecular nanostructure and stability of the nanofiber. Improved hemocompatibility requires PEGylation on stable supramolecular nanofibers composed of a highly organized β-sheet structure while PEG conjugation on weakly packed nanofibers composed of partially denatured β-sheets did not show improvement. The current study reveals the fundamental mechanism involved in the selective hemocompatibility improvement of the SAANs upon PEG conjugation. The structure–activity relationship developed in this study will provide important guidance for the future design of a broader family of peptide and polymer-based assemblies with optimized antimicrobial activity and hemocompatibility.

International audience; Chloroquine (CQ) has been widely used in the treatment of malaria since the 1950s, though toxicity and resistance is increasingly limiting its use in the clinic. More recently, CQ is also becoming recognized as an important therapeutic compound for the treatment of autoimmune disorders and has shown activity as an anticancer agent. However, the full extent of CQ pharmacology in humans is still unclear. Herein, we demonstrate that the lysosomal protein saposinB (sapB), critical for select lipid degradation, binds CQ with implications for both CQ function and toxicity. Using isothermal titration calorimetry (ITC) and fluorescence quenching experiments, CQ was shown to bind to the dimeric form of sapB at both pH5.5 and pH7.4 with an average binding affinity of 2.3x10(4)m(-1). X-ray crystallography confirmed this, and the first complete crystal structure of sapB with a bound small molecule (CQ) is reported. The results suggest that sapB might play a role in mitigating CQ-based toxicity and that sapB might itself be overwhelmed by CQ causing impaired lipid degradation.

Transferrin (TF) is a bilobal transport protein that acquires ferric iron from the diet and holds it tightly within the cleft of each lobe (thereby preventing its hydrolysis). The iron is delivered to actively dividing cells by receptor mediated endocytosis in which diferric TF preferentially binds to TF receptors (TFRs) on the cell surface and the entire complex is taken into an acidic endosome. A combination of lower pH, a chelator, inorganic anions, and the TFR leads to the efficient release of iron from each lobe. Identification of residues/regions within both TF and TFR required for high affinity binding has been an ongoing goal in the field. In the current study, we created human TF (hTF) mutants to identify a region critical to the interaction with the TFR which also constitutes part of an overlapping epitope for two monoclonal antibodies (mAbs) to the N-lobe, one of which was previously shown to block binding of hTF to the TFR. Four single point mutants, P142A, R143A, K144A, and P145A in the N-lobe, were placed into diferric hTF. Isothermal titration calorimetry (ITC) revealed that three of the four residues (Pro142, Lys144, and Pro145) in this loop are essential to TFR binding. Additionally, Lys144 is common to the recognition of both mAbs which show different sensitivities to the three other residues. Taken together these studies prove that this loop is required for binding of the N-lobe of hTF to the TFR, provide a more precise description of the role of each residue in the loop in the interaction with the TFR, and confirm that the N-lobe is essential to high affinity binding of diferric hTF to TFR. Copyright © 2009 John Wiley & Sons, Ltd.

Background: The acetylcholine activated inward rectifier potassium current (IKACh) was shown to be constitutively active in chronic atrial fibrillation. Its blockade has been proposed to be a possible antifibrillatory pharmacotherapy. We therefore tested the hypothesis that blocking IKACh with the bee venom peptide tertiapin, or the small molecule chloroquine, terminates chronic AF in the sheep heart. Methods and Results: We tested our hypothesis using biochemistry, electrophysiology and molecular modeling. In patch clamp, the IC50 of IKACh block by tertiapin, and chloroquine was 60 nM, and 710 nM respectively, while dofetilde, a currently used class III antiarrhythmic, did not block the current. On the other hand, dofetilide and chloroquine blocked the rapid delayed rectifier potassium current (IKr) with an IC50 of 50nM, and 2.3 microM respectively, while tertiapine had no effects. Furthermore, molecular modeling indicated that 1 chloroquine molecule blocks the intracellular ion permeation vestibule of the tetrameric Kir3.1, a molecular correlate of IKACh, by interacting with amino acids important for the channel’s rectification. Dofetilide did not interact with Kir3.1. In vitro fluorescence measurements of chloroquine titration into the purified Kir3.1 intracellular domain protein confirmed the computational results and indicated that chloroquine directly binds Kir3.1 with a stoichiometry of 1 chloroquine molecule per Kir3.1 tetramer. Finally, optical mapping of the chronically fibrillating sheep atria showed that while tertiapine, chloroquine, and dofetilide slowed down chronic AF and prolonged the action potential, only tertiapine and chloroquine (the IKACh blockers) restored normal sinus rhythm. Conclusions: IKACh could be a more powerful anti atrial fibrillation target compared to IKr. Pharmacological blockade of IKACh with a peptide, or a small molecule similar to chloroquine could terminate chronic AF and restore normal sinus rhythm.

INRA, MIG, Domaine de Vilvert, F-78352 Jouy en Josas, France : depuis janvier 2015 : INRA, MaIAGE, Domaine de Vilvert, F-78352 Jouy en Josas, France; Iron is an essential element for bacterial growth and virulence. Because of its limited bioavailability in the host, bacteria have adapted several strategies to acquire iron during infection. In the human opportunistic bacteria Bacillus cereus, a surface protein IlsA is shown to be involved in iron acquisition from both ferritin and hemoproteins. IlsA has a modular structure consisting of a NEAT (Near Iron transporter) domain at the N-terminus, several LRR (Leucine Rich Repeat) motifs and a SLH (Surface Layer Homology) domain likely involved in anchoring the protein to the cell surface.METHODS:[b]BACKGROUND:[/b]Isothermal titration calorimetry, UV-Vis spectrophotometry, affinity chromatography and rapid kinetics stopped-flow measurements were employed to probe the binding and transfer of hemin between two different B. cereus surface proteins (IlsA and IsdC).[br/][b]RESULTS:[/b]IlsA binds hemin via the NEAT domain and is able to extract heme from hemoglobin whereas the LRR domain alone is not involved in these processes. A rapid hemin transfer from hemin-containing IlsA (holo-IlsA) to hemin-free IsdC (apo-IsdC) is demonstrated.[br/][b]CONCLUSIONS[/b]:For the first time, it is shown that two different B. cereus surface proteins (IlsA and IsdC) can interact and transfer heme suggesting their involvement in B. cereus heme acquisition.[br/][b]GENERAL SIGNIFICANCE:[/b]An important role for the complete Isd system in heme-associated bacterial growth is demonstrated and new insights into the interplay between an Isd NEAT surface protein and an IlsA-NEAT-LRR protein, both of which appear to be involved in heme-iron acquisition in B. cereus are revealed.

From microorganisms to humans, ferritin plays a central role in the biological management of iron. The ferritins function as iron storage and detoxification proteins by oxidatively depositing iron as a hydrous ferric hydroxide mineral core within their shell-like structures. The mechanism by which the mineral core is formed has been the subject of intense investigation for many years. A diiron ferroxidase site located on the H-chain subunit of vertebrate ferritins catalyzes the oxidation of Fe(II) to Fe(III) by molecular oxygen. A previous stopped-flow kinetics study of a transient mu-peroxodiFe(III) intermediate formed at this site revealed very unusual kinetics curves, the shape of which depended markedly on the amount of iron presented to the protein. In the present work, a mathematical model for catalysis is developed that explains the observed kinetics. The model consists of two sequential mechanisms. In the first mechanism, turnover of iron at the ferroxidase site is rapid, resulting in steady-state production of the peroxo intermediate with continual formation of the mineral core until the available Fe(II) in solution is consumed. At this point, the second mechanism comes into play whereby the peroxo intermediate decays and the ferroxidase site is postulated to vacate its complement of iron. The kinetic data reveal for the first time that Fe(II) in excess of that required to saturate the ferroxidase site promotes rapid turnover of Fe(III) at this site and that the ferroxidase site plays a role in catalysis at all levels of iron loading of the protein (48-800 Fe/protein). The data also provide evidence for a second intermediate, a putative hydroperoxodiFe(III) complex, that is a decay product of the peroxo intermediate.

Bacterioferritin (EcBFR) ofEscherichia coli is an iron-mineralizing hemoprotein composed of 24 identical subunits, each containing a dinuclear metal-binding site known as the “ferroxidase center.” The chemistry of Fe(II) binding and oxidation and Fe(III) hydrolysis using H2O2 as oxidant was studied by electrode oximetry, pH-stat, UV-visible spectrophotometry, and electron paramagnetic resonance spin trapping experiments. Absorption spectroscopy data demonstrate the oxidation of two Fe(II) per H2O2 at the ferroxidase center, thus avoiding hydroxyl radical production via Fenton chemistry. The oxidation reaction with H2O2 corresponds to [Fe(II)2-P]Z + H2O2→ [Fe(III)2O-P]Z + H2O, where [Fe(II)2-P]Z represents a diferrous ferroxidase center complex of the protein P with net charge Z and [Fe(III)2O-P]Z a μ-oxo-bridged diferric ferroxidase complex. The mineralization reaction is given by 2Fe2+ + H2O2 + 2H2O → 2FeOOH(core) + 4H+, where two Fe(II) are again oxidized by one H2O2. Hydrogen peroxide is shown to be an intermediate product of dioxygen reduction when O2 is used as the oxidant in both the ferroxidation and mineralization reactions. Most of the H2O2produced from O2 is rapidly consumed in a subsequent ferroxidase reaction with Fe(II) to produce H2O. EPR spin trapping experiments show that the presence of EcBFR greatly attenuates the production of hydroxyl radical during Fe(II) oxidation by H2O2, consistent with the ability of the bacterioferritin to facilitate the pairwise oxidation of Fe(II) by H2O2, thus avoiding odd electron reduction products of oxygen and therefore oxidative damage to the protein and cellular components through oxygen radical chemistry.

Vitamin A based bisretinoid accumulation is a major focus in the study of macular degeneration. Whether specific endogenous lysosomal proteins can bind A2E, a pronounced bisretinoid in lipofuscin granules in retinal pigment epithelial cells, and interfere with enzymatic or photoinduced oxidation of such, has not been explored. Herein, using fluorescence and electronic absorption spectroscopy and mass spectrometry, we demonstrate that Saposin B, a critical protein in the degradation of sulfatides and “flushing” of lipids, can bind A2E, preventing its H2O2-dependent enzymatic oxidation by horseradish peroxidase and photooxidation by blue light (λ=450–460 nm).

Tridentate chelate ligands of 2,6-bis[hydroxy(methyl)amino]-1,3,5-triazine family rapidly release iron from human recombinant ferritin in the presence of oxygen. The reaction is inhibited by superoxide dismutase, catalase, mannitol and urea. Suggested reaction mechanism involves reduction of the ferritin iron core by superoxide anion, diffusion of iron(II) cations outside the ferritin shell, and regeneration of superoxide anions through oxidation of iron(II) chelate complexes with molecular oxygen.

Iron uptake by transferrin from triacetohydroxamatoFe(III) (Fe(AHA)3) in the presence of bicarbonate has been investigated between pH 7 and 8.2. The protein transits from the opened apo- to the closed holoform by several steps with the accumulation of at least three kinetic intermediates. All these steps are accompanied by proton losses, probably occurring from the protein ligands and the side-chains involved in the interdomain H-bonding nets. The minor bihydroxamatoFe(III) species Fe(AHA)2 exchanges its iron with the C-site of apotransferrin in interaction with bicarbonate without detectable formation of any intermediate protein-iron-ligand mixed complex; direct second-order rate constant k1 = 4.15(±0.05) × 107 M−1 s−1. The kinetic product loses a single proton and undergoes a modification in its conformation followed by the loss of two or three protons; first-order rate constant k2 = 3.25(±0.15) s−1. This induces a new modification in the conformation; first-order rate constant k3 = 5.90(±0.30) × 10−2 s−1. This new modification in conformation rate controls iron uptake by the N-site of the protein and is followed by a single proton loss; K3a = 6.80 nM. Finally, the holoprotein or the monoferric transferrin in its thermodynamic equilibrated state is produced by a last modification in the conformation occurring in about 4000 seconds. But for the Fe(AHA)3 dissociation and the involvement of Fe(AHA)2 in the first step of iron uptake, this mechanism is identical to that reported for iron uptake from FeNAc3. This implies that the exchange of iron between a chelate and serum-transferrin occurs by a single general mechanism. The nature of the iron-providing chelate is only important for the first kinetic step of the exchange, which can be slowed to such an extent that it rate limits the exchange of iron.

Iron release from ovotransferrin in acidic media (3pH6) occurs in at least six kinetic steps. The first is a very fast (/= 5 ms) decarbonation of the iron-loaded protein. Iron release from both sites of the protein is controlled by what appear to be slow proton transfers. The N-site loses its iron first in two steps, the first occurring in the tenth of a second range with second order rate constant k1 = (2.30 +/- 0.10) x 104 M-1.s-1, first order rate constant k-1 = (1.40 +/- 0.10) s-1 and equilibrium constant K1a = (60 +/- 6) microM. The second step occurs in the second range with a second order rate constant k2 = (5.2 +/- 0.15) x 103 M-1.s-1, first order rate constant k-2 = (0.2 +/- 0.02) s-1 and equilibrium constant K2a = (39 +/- 5) microM. Iron is afterward lost from the C-site of the protein by two different pathways, one in the presence of a strong Fe(III) ligand such as citrate and the other in the presence of weak ligands such as formate or acetate. The first step, common to both paths, is a slow proton uptake which occurs in the tens of second range with a second order rate constant k3 = (1.22 +/- 0.03) x 103 M-1.s-1 and equilibrium constant K3a = (1.0 +/- 0.1) mM. In the presence of citrate, this step is followed by formation of an intermediate complex with monoferric ovotransferrin; stability constant KLC = (0.435 +/- 0.015) mM. This last step is rate-controlled by slow proton gain which occurs in the hundred second range with a second order rate constant k4 = (1.05 +/- 0.05) x 104 M-1.s-1, first order rate constant k-4 = (1.0 +/- 0.1) x 10-2 s-1 and equilibrium constant K4a = (0.95 +/- 0.15) microM. In the presence of a weak iron(III) ligand such as acetate or formate, formation of an intermediate complex is not detected and iron release is controlled by two final slow proton uptakes. The first occurs in the hundred to thousand second range, second order rate constant k5 = (6.90 +/- 0.30) x 106 M-1.s-1. The last step occurs in the thousand second range. Iron release by ovotransferrin is similar but not identical to that of serum-transferrin. It is slower and occurs at lower pH values. However, as seen for serum-transferrin, it seems to involve the protonation of the amino acid side-chains involved in iron co-ordination and perhaps those implicated in interdomain H-bonds. The observed proton transfers are, then, probably controlled by the change in conformation of the binding lobes from closed when iron-loaded to open in the apo-form.

Ferritins are ubiquitous iron mineralizing and storage proteins that play an important role in iron homeostasis. Although excess iron is stored in the cytoplasm, most of the metabolically active iron is processed in the mitochondria of the cell. Little is known about how these organelles regulate iron homeostasis and toxicity. The recently discovered human mitochondrial ferritin (MtF), unlike other mammalian ferritins, is a homopolymer of 24 subunits that has a high degree of sequence homology with human H-chain ferritin (HuHF). Parallel experiments with MtF and HuHF reported here reveal striking differences in their iron oxidation and hydrolysis chemistry despite their similar diFe ferroxidase centers. In contrast to HuHF, MtF does not regenerate its ferroxidase activity after oxidation of its initial complement of Fe(II) and generally has considerably slower ferroxidation and mineralization activities as well. MtF exhibits sigmoidal kinetics of mineralization more characteristic of an L-chain than an H-chain ferritin. Site-directed mutagenesis reveals that serine 144, a residue situated near the ferroxidase center in MtF but absent from HuHF, is one player in this impairment of activity. Additionally only one-half of the 24 ferroxidase centers of MtF are functional, further contributing to its lower activity. Stopped-flow absorption spectrometry of Fe(II) oxidation by O(2) in MtF shows the formation of a transient diiron(III) mu-peroxo species (lambda(max) = 650 nm) as observed in HuHF. Also, as for HuHF, minimal hydroxyl radical is produced during the oxidative deposition of iron in MtF using O(2) as the oxidant. However, the 2Fe(II) + H(2)O(2) detoxification reaction found in HuHF does not occur in MtF. The structural differences and the physiological implications of the unique iron oxidation properties of MtF are discussed in light of these results.

Human serum transferrin (hTf) is a single-chain bilobal glycoprotein that efficiently delivers iron to mammalian cells by endocytosis via the transferrin/transferrin receptor system. While extensive studies have been directed towards the study of ferric ion binding to hTf, ferrous ion interactions with the protein have never been firmly investigated owing to the rapid oxidation of Fe(II) to Fe(III) and the difficulty in maintaining a fully anaerobic environment. Here, the binding of Fe(2+) and Zn(2+) ions to hTf has been studied under anaerobic and aerobic conditions, respectively, in the presence and absence of bicarbonate by means of isothermal titration calorimetry (ITC) and fluorescence spectroscopy. The ITC data indicate the presence of one class of strong binding sites with dissociation constants of 25.2 nM for Fe(2+) and 6.7 nM for Zn(2+) and maximum binding stoichiometries of 1 Zn(2+) (or 1 Fe(2+)) per hTf molecule. With either metal, the binding interaction was achieved by both favorable enthalpy and entropy changes (ΔH(0)~-12 kJ/mol and ΔS(0)~106 J/mol·K for Fe(2+) and ΔH(0)~-18 kJ/mol and ΔS(0)~97 J/mol·K for Zn(2+)). The large and positive entropy values are most likely due to the change in the hydration of the protein and the metal ions upon interaction. Rapid kinetics stopped-flow fluorescence spectroscopy revealed two different complexation mechanisms with different degrees of conformational changes upon metal ion binding. Our results are discussed in terms of a plausible scenario for iron dissociation from transferrin by which the highly stable Fe(3+)-hTf complex might be reduced to the more labile Fe(2+) ion before iron is released to the cytosol.

Background Extensive in-vitro studies have focused on elucidating the mechanism of iron uptake and mineral core formation in ferritin. However, despite a plethora of studies attempting to characterize iron release under different experimental conditions, the in-vivo mobilization of iron from ferritin remains poorly understood. Several iron-reductive mobilization pathways have been proposed including, among others, flavin mononucleotides, ascorbate, glutathione, dithionite, and polyphenols. Here, we investigate the kinetics of iron release from ferritin by reduced flavin nucleotide, FMNH2, and discuss the physiological significance of this process in-vivo. Methods Iron release from horse spleen ferritin and recombinant human heteropolymer ferritin was followed by the change in optical density of the Fe(II)–bipyridine complex using a Cary 50 Bio UV–Vis spectrophotometer. Oxygen consumption curves were followed on a MI 730 Clark oxygen microelectrode. Results The reductive mobilization of iron from ferritin by the nonenzymatic FMN/NAD(P)H system is extremely slow in the presence of oxygen and might involve superoxide radicals, but not FMNH2. Under anaerobic conditions, a very rapid phase of iron mobilization by FMNH2 was observed. Conclusions Under normoxic conditions, FMNH2 alone might not be a physiologically significant contributor to iron release from ferritin. General significance There is no consensus on which iron release pathway is predominantly responsible for iron mobilization from ferritin under cellular conditions. While reduced flavin mononucleotide (FMNH2) is one likely candidate for in-vivo ferritin iron removal, its significance is confounded by the rapid oxidation of the latter by molecular oxygen.

Background In mammals, serum-transferrins transport iron from the neutral environment of the blood to the cytoplasm by receptor-mediated endocytosis. Extensive in-vitro studies have focused on the thermodynamics and kinetics of Fe3+ binding to a number of transferrins. However, little attention has been given to the thermodynamic characterization of the interaction of transferrin with its receptor. Scope of review Iron-loaded transferrin (Tf) binds with high affinity to the specific transferrin receptor (TfR) on the cell surface. The Tf–TfR complex is then internalized via receptor mediated endocytosis into an endosome where iron is released. Here, we provide an overview of recent studies that have used ITC to quantify the interaction of various metal ions with transferrin and highlight our current understanding of the thermodynamics of the transferrin–transferrin receptor system at physiological pH. General significance The interaction of the iron-loaded transferrin with the transferrin receptor is a key cellular process that occurs during the normal course of iron metabolism. Understanding the thermodynamics of this interaction is important for iron homeostasis since the physiological requirement of iron must be appropriately maintained to avoid iron-related diseases. Major conclusions The thermodynamic data revealed stoichiometric binding of all tested metal ions to transferrin with very high affinities ranging between 1017 and 1022 M− 1. Iron-loaded transferrin (monoferric or diferric) is shown to bind avidly (K ~ 107–108 M− 1) to the receptor at neutral pH with a stoichiometry of one Tf molecule per TfR monomer. Significantly, both the N- and the C-lobe contribute to the binding interaction which is shown to be both enthalpically and entropically driven. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.