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Abstract

The apparent affinity of human serum albumin (HSA) for divalent copper has long been the subject of great interest, due to its presumed role as the major Cu2+ -binding ligand in blood and cerebrospinal fluid. Using a combination of electronic absorption, circular dichroism and room-temperature electron paramagnetic resonance spectroscopies, together with potentiometric titrations, we competed the tripeptide GGH against HSA to reveal a conditional binding constant of log c K Cu Cu ( HSA ) =13.02±0.05 at pH 7.4. This rigorously determined value of the Cu2+ affinity has important implications for understanding the extracellular distribution of copper.

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... [16] For instance, Cu II binds to HSA motif (Asp-Ala-His-, DAH-) with log c K7.4 = 13, while it binds to Neuromedin C, whose Gly-Asn-His-Trp (GNHW) motif was adopted in the probe GNH W/Tb , with log c K7.4 = 13.6. [17,18] Cu II -XZH complexes are stable over a large pH range, classically from above pH 10 down to about 5.5, where decoordination starts to occur. [19][20][21] Importantly, Zn II does not seem able to bind to the ATCUN motif (at least at pH < 9), due to its lower Lewis acid character (crucial to favour the deprotonation of the two amide groups) and the preference for tetrahedral rather than planar coordination geometry. ...
... [19] Instead, Cu II -AHH W/Tb and Cu II -HSA could be discriminated at room temperature (RT), where different EPR spectra can be obtained for complexes with the same coordination mode but different tumbling rate (Fig. 4B). [17] After incubation of Cu II -HSA with AHH W/Tb , the mixture showed an RT-EPR spectrum (Fig. 4B, green curve) very similar to that of Cu II -AHH W/Tb (red curve), confirming that AHH W/Tb retrieves Cu II from HSA (as observed by luminescence, Fig. 3A). Besides, the LT-EPR spectrum recorded after the Cu II transfer to AHH W/Tb (Fig. 4A, green curve) was indistinguishable (within experimental error) from that of Cu II -AHH W/Tb (red curve). ...
Article
The measurement of labile CuII in biological samples is fundamental for understanding Cu metabolism and has been emerging as a promising diagnostic marker for Cu-related pathologies such as Wilson's and Alzheimer's diseases. The use of fluorescent chelators may be useful to circumvent separation steps employed by current methods. For this purpose, we recently designed a selective and suited-affinity turn-off luminescent probe based on a peptide bearing the CuII-binding Xxx-Zzz-His (Amino-Terminal CuII- and NiII-binding, ATCUN) motif and a TbIII-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) complex. Here, we present an analogue probe bearing the ATCUN motif variant Xxx-His-His. This probe showed much faster response in biologically-relevant media and higher stability than the previous motif at low pH. These features could be beneficial to the measurement of dynamic CuII fluctuations and the application in slightly acidic media, such as urine.
... Ceruloplasmin (132 kDa) Ferroxidase 0,2-0.5g/L (~2.6 µM) [14] ~ 90% (6) [14] 65-71% [23] 95% [14] (~10-15 µM) n/a Inert [12] Serum Albumin (66 kDa) Carrier (fatty acids, metal ions, …) ~ 0.6 mM < 2% (1) [24] 15-19% (~3µM) [23] < 5% [14] 13 [18] t1/2, ON < 1 sec [19,20] t1/2, OFF ~ min [12,25] α2-Macroglobulin (725 kDa) ...
... Herein, Cu 2+ MRI probes reported to date are described and discussed (see Table 11 and Figure 9). In 2006, Chang et al. presented the first Cu 2+ -activated MRI contrast agent (18) and a few years later, they reported other Cu + -and a Cu 2+ -responsive CAs (19). [254,255] Both Cu 2+ -activated CAs contain a Gd 3+ -DO3A complex linked to a Cu 2+binding group. ...
Article
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Copper (Cu) is an essential micronutrient for most organisms and serves mainly as a redox-active catalytic centre in enzymes cycling between Cu+ and Cu2+. Membrane transporters and shuttles are involved to bring and insert Cu into these enzymes and to control tightly the copper metabolism, whose failure can lead to severe diseases. The main oxidation state intracellularly is Cu+, whereas Cu2+ is mainly found in extracellular fluids. A basic approach to investigate Cu metabolism in vivo and in cellulo contemplates the use of luminescent (mostly fluorescent) or magnetic resonance imaging (MRI)-active probes. Here, we focus on sensors of the Cu2+ state. First, Cu metabolism and speciation are revised, focusing on the main extracellular fluids (blood plasma, urine, cerebrospinal fluid, synaptic cleft, milk, saliva, sweat) and cell culture media, and highlighting the notion of exchangeable Cu2+ pool. Indeed, in contrast to bulk Cu measurements, sensors can only detect Cu2+ that is labile and thermodynamically accessible. Thus, the kinetics and thermodynamics of the exchangeable pools determine the quantity of Cu2+ that can be measured and influence the design of the sensor. The study of the best-known exchangeable Cu2+ pool in blood plasma, i.e. serum albumin, shows that a sensor might need a sub-femtomolar affinity for Cu2+ to compete with endogenous Cu2+ ligands. The selectivity of the probe for Cu2+ is also discussed, in particular against Zn2+, which is much more available than Cu2+ in the extracellular fluids (e.g. at least 106 times in the blood). Finally, the analysis of the literature on luminescent and MRI-active Cu2+ sensors applied in extracellular media show indeed how challenging such measurement is, and that none of the sensors reported convincingly and specifically detects Cu2+ in a biological system. Indeed, when considering all the sought parameters, i.e. thermodynamics and kinetics of the Cu2+-sensor, the specificity towards Cu2+, the reversibility, the sensitivity of the luminescent or MRI response and hence the required sensor concentration, it becomes clear that this is a huge challenge and that we stand just at the dawn of this field.
... The relaxivity increase is amplified to 270% in the presence of HSA. Cu 2+ is bound to the N-terminal site of HSA [71] with a log KCu = 13 [72], suggesting that Cu 2+ remains in this domain of the protein in the presence of GdP33. This is confirmed by XANES experiments, showing very similar signatures of Cu-HSA and GdP33-Cu-HSA, supporting the fact that Cu 2+ remains in the same environment whether GdP33 is present or not. ...
... The relaxivity increase is amplified to 270% in the presence of HSA. Cu 2+ is bound to the N-terminal site of HSA [71] with a log K Cu = 13 [72], suggesting that Cu 2+ remains in this domain of the protein in the presence of GdP33. This is confirmed by XANES experiments, showing very similar signatures of Cu-HSA and GdP33-Cu-HSA, supporting the fact that Cu 2+ remains in the same environment whether GdP33 is present or not. ...
Article
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Zinc and copper are essential cations involved in numerous biological processes, and variations in their concentrations can cause diseases such as neurodegenerative diseases, diabetes and cancers. Hence, detection and quantification of these cations are of utmost importance for the early diagnosis of disease. Magnetic resonance imaging (MRI) responsive contrast agents (mainly Lanthanide(+III) complexes), relying on a change in the state of the MRI active part upon interaction with the cation of interest, e.g., switch ON/OFF or vice versa, have been successfully utilized to detect Zn2+ and are now being developed to detect Cu2+. These paramagnetic probes mainly exploit the relaxation-based properties (T1-based contrast agents), but also the paramagnetic induced hyperfine shift properties (paraCEST and parashift probes) of the contrast agents. The challenges encountered going from Zn2+ to Cu2+ detection will be stressed and discussed herein, mainly involving the selectivity of the probes for the cation to detect and their responsivity at physiologically relevant concentrations. Depending on the response mechanism, the use of fast-field cycling MRI seems promising to increase the detection field while keeping a good response. In vivo applications of cation responsive MRI probes are only in their infancy and the recent developments will be described, along with the associated quantification problems. In the case of relaxation agents, the presence of another method of local quantification, e.g., synchrotron X-Ray fluorescence, single-photon emission computed tomography (SPECT) or positron emission tomography (PET) techniques, or 19F MRI is required, each of which has its own advantages and disadvantages.
... [5] There is, however, at least one thoroughly characterized linear Cu(II)-binding motif engaging threeamino-acid N-terminal sequence, with histidine residue in the third position (His 3 ), amino terminal Cu and Ni (ATCUN) motif. The examples of such sequences are: Asp-Ala-His in human serum albumin (HSA), [6] and Phe-Arg-His in N-truncated human amyloid beta (Aβ) peptide. [7] Both these sequences bind Cu(II) ions strongly, with femtomolar dissociation constants at pH 7.4. ...
... [7] Both these sequences bind Cu(II) ions strongly, with femtomolar dissociation constants at pH 7.4. [6,7] Interaction of Cu(II) with proteins comprising such sequences is biologically relevant as, e. g., 10 -15 % of plasma copper is kept in the ATCUN motif of HSA. [8] Interestingly, peptides/proteins with histidine residue in the second position of the amino acid sequence (His 2 ) also provide excellent coordination ligands for Cu(II), as was shown for Gly-His-Lys peptide, the wound healing factor. ...
Article
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Link to Free Full Text: ............................................................................................................................................ https://onlinelibrary.wiley.com/share/author/J5YPUTRMDWJNMENJYC8K?target=10.1002/cbdv.202100043 ............................................................................................................................................ Proteins anchor copper(II) ions mainly by imidazole from histidine residues located in different positions in the primary protein structures. However, the motifs with histidine in the first three N-terminal positions (His1, His2, and His3) show unique Cu(II)-binding properties, such as availability from the surface of the protein, high flexibility, and high Cu(II) exchangeability with other ligands. It makes such sequences beneficial for the fast exchange of Cu(II) between ligands. Furthermore, sequences with His1 and His2, thus, non-saturating the Cu(II) coordination sphere, are redox-active and may play a role in Cu(II) reduction to Cu(I). All human protein sequences deposited in UniProt Knowledgebase were browsed for those containing His1, His2, or His3. Proteolytically modified sequences (with the removal of a propeptide or Met residue) were taken for the analysis. Finally, the sequences were sorted out according to the subcellular localization of the proteins to match the respective sequences with the probability of interaction with divalent copper.
... 13 However, many of these studies are limited to structural studies with isolated proteins in buffer, snapshots of copper localization in xed tissues and cells, or indirect measures via tracking of copper-dependent proteins and enzymes. [14][15][16][17] The ability to target and monitor the copper pools in both the intracellular and extracellular milieu would provide valuable insight into copper dynamics in both normal physiological and pathological states. ...
Article
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Copper is an essential redox-active metal that plays integral roles in biology ranging from enzymatic catalysis to mitochondrial respiration. However, if not adequately regulated, this redox activity has the potential to cause oxidative stress through the production of reactive oxygen species. Indeed, the dysregulation of copper has been associated with a variety of disease states including diabetes, neurodegenerative disorders, and multiple cancers. While increasing tools are being developed for illuminating labile intracellular copper pools and the trafficking pathways in which they are involved, significantly less attention has been given to the analogous extracellular labile pool. To address this gap, we have developed a bioluminescence-based imaging probe, picolinic ester caged-diphenylterazine (pic-DTZ) for monitoring labile, extracellular copper using a coelenterazine-like imidazopyrazinone and the genetically-engineered, marine-based luciferase, nanoluciferase. Unlike the more commonly-used firefly luciferase, nanoluciferase does not require ATP, allowing its application to the extracellular milieu. pic-DTZ demonstrates high metal and oxidation state selectivity for Cu(ii) in aqueous buffer as well as selectivity for labile pools over coordinatively inaccessible protein-bound Cu(ii). We demonstrate the potential of pic-DTZ as a diagnostic tool in human serum and plasma for copper-associated diseases. Additionally, we apply pic-DTZ to lend insight into the extracellular copper dynamic in anticancer treatments.
... Cu(II)-binding properties of NTS have been in the focus of intensive studies. The dissociation constant (K D ) values for Cu(II) in NTS of HSA (Asp-Ala-His) and of bovine serum albumin (Asp-Thr-His), have been determined by a variety of methods such as potentiometric titration, equilibrium dialysis, ultrafiltration, isothermal titration calorimetry, Cu(II) electrode and several spectroscopic methods, including EPR 27 . However, the estimated values for the conditional dissociation constant vary from picomolar (log K D = −11.18) to subfemtomolar (log K D = −16.18) ...
Article
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It has been reported that Cu(II) ions in human blood are bound mainly to serum albumin (HSA), ceruloplasmin (CP), alpha-2-macroglobulin (α2M) and His, however, data for α2M are very limited and the thermodynamics and kinetics of the copper distribution are not known. We have applied a new LC-ICP MS-based approach for direct determination of Cu(II)-binding affinities of HSA, CP and α2M in the presence of competing Cu(II)-binding reference ligands including His. The ligands affected both the rate of metal release from Cu•HSA complex and the value of KD. Slow release and KD = 0.90 pM was observed with nitrilotriacetic acid (NTA), whereas His showed fast release and substantially lower KD = 34.7 fM (50 mM HEPES, 50 mM NaCl, pH 7.4), which was explained with formation of ternary His•Cu•HSA complex. High mM concentrations of EDTA were not able to elicit metal release from metallated CP at pH 7.4 and therefore it was impossible to determine the KD value for CP. In contrast to earlier inconclusive evidence, we show that α2M does not bind Cu(II) ions. In the human blood serum ~75% of Cu(II) ions are in a nonexchangeable manner bound to CP and the rest exchangeable copper is in an equilibrium between HSA (~25%) and Cu(II)-His-Xaa ternary complexes (~0.2%).
... This peptide, together with its non-amidated counterpart, is the simplest ATCUN model, with amidation providing a better mimic of the peptide chain extension in ATCUN peptides/proteins. 31,59 The GSH addition to Cu(II)GGH caused a partial Cu(II) reduction manifested by a decrease of the d−d band at 525 nm ( Figure S30B), analogous to Cu(II)GHK and Im-Cu(II)GHK, but about 50-fold slower ( Figure 5). In turn, the timespan of re-oxidation was similar for all the three systems, about 1 hour in our experimental conditions. ...
Article
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Gly-His-Lys (GHK) is a tripeptide present in the human bloodstream that exhibits a number of biological functions. Its activity is attributed to the copper-complexed form, Cu(II)GHK. Little is known, however, about the molecular aspects of the mechanism of its action. Here, we examined the reaction of Cu(II)GHK with reduced glutathione (GSH), which is the strongest reductant naturally occurring in human plasma. Spectroscopic techniques (UV–vis, CD, EPR, and NMR) and cyclic voltammetry helped unravel the reaction mechanism. The impact of temperature, GSH concentration, oxygen access, and the presence of ternary ligands on the reaction were explored. The transient GSH-Cu(II)GHK complex was found to be an important reaction intermediate. The kinetic and redox properties of this complex, including tuning of the reduction rate by ternary ligands, suggest that it may provide a missing link in copper trafficking as a precursor of Cu(I) ions, for example, for their acquisition by the CTR1 cellular copper transporter.
... 16,17 Two N-terminal sequence motifs, Xaa-His and Xaa-Zaa-His (where Xaa is any α-amino acid except of Cys, and Zaa is any α-amino acid except of Cys or Pro), provide the highest Cu(II) complex affinities by virtue of synergistic formation of chelate rings involving peptide nitrogen atoms ( Figure 1). 18−20 The logarithmic conditional stability constants at physiological pH 7.4, log C K 7.4 , for Xaa-His complexes are in the range of 12.5−13, while those of Xaa-Zaa-His complexes range from 12.3 to ca. 15. 21,22 The latter are also known as ATCUN or NTS complexes. 18 Mass spectrometry (MS) in its many variants is one of the most versatile techniques of peptide and protein research, providing information on their composition, sequence, and post-translational modifications and via hyphenated techniques also on protein structure. ...
Article
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The toolset of mass spectrometry (MS) is still expanding, and the number of metal ion complexes researched this way is growing. The Cu(II) ion forms particularly strong peptide complexes of biological interest which are frequent objects of MS studies, but quantitative aspects of some reported results are at odds with those of experiments performed in solution. Cu(II) complexes are usually characterized by fast ligand exchange rates, despite their high affinity, and we speculated that such kinetic lability could be responsible for the observed discrepancies. In order to resolve this issue, we selected peptides belonging to the ATCUN family characterized with high and thoroughly determined Cu(II) binding constants and re-estimated them using two ESI-MS techniques: standard conditions in combination with serial dilution experiments and very mild conditions for competition experiments. The sample acidification, which accompanies the electrospray formation, was simulated with the pH–jump stopped-flow technique. Our results indicate that ESI-MS should not be used for quantitative studies of Cu(II)–peptide complexes because the electrospray formation process compromises the entropic contribution to the complex stability, yielding underestimations of complex stability constants.
... Baseline correction was performed by weighted subtraction of the spectrum obtained using a water blank. Spectral simulations were carried out as previously described [51]. ...
Article
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The tripeptide NH2–Gly–His–Lys–COOH (GHK), cis-urocanic acid (cis-UCA) and Cu(II) ions are physiological constituents of the human body and they co-occur (e.g., in the skin and the plasma). While GHK is known as Cu(II)-binding molecule, we found that urocanic acid also coordinates Cu(II) ions. Furthermore, both ligands create ternary Cu(II) complex being probably physiologically functional species. Regarding the natural concentrations of the studied molecules in some human tissues, together with the affinities reported here, we conclude that the ternary complex [GHK][Cu(II)][cis-urocanic acid] may be partly responsible for biological effects of GHK and urocanic acid described in the literature.
... The spectroscopic readout may include signal derived from UV-visible absorbance, fluorescence, circular dichroism, electron paramagnetic resonance (EPR) or nuclear magnetic resonance (NMR) [11,43]. The readout from a single species is usually enough for quantification of the binding equilibrium such as equations 3, 7a or 7b but it is sometimes possible, and may increase robustness, to detect multiple species in equilibrium simultaneously [52]. In the next section, we review in detail the application of some commonly used spectroscopic probes in metal-protein affinity determination. ...
Article
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Metal ions play many critical roles in biology, as structural and catalytic cofactors, and as cell regulatory and signalling elements. The metal–protein affinity, expressed conveniently by the metal dissociation constant, KD, describes the thermodynamic strength of a metal–protein interaction and is a key parameter that can be used, for example, to understand how proteins may acquire metals in a cell and to identify dynamic elements (e.g. cofactor binding, changing metal availabilities) which regulate protein metalation in vivo. Here, we outline the fundamental principles and practical considerations that are key to the reliable quantification of metal–protein affinities. We review a selection of spectroscopic probes which can be used to determine protein affinities for essential biological transition metals (including Mn(II), Fe(II), Co(II), Ni(II), Cu(I), Cu(II) and Zn(II)) and, using selected examples, demonstrate how rational probe selection combined with prudent experimental design can be applied to determine accurate KD values.
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The measurement of exchangeable Cu2+ levels in biological samples is gaining interest in the context of copper-related pathologies. Here, we report a Tb3+ luminescent turn-off sensor for Cu2+ based on...
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The amino‐terminal copper and nickel/N‐terminal site (ATCUN/NTS) present in proteins and bioactive peptides exhibits high affinity towards Cu II ions and have been implicated in human copper physiology. Little is known, however, about the rate and exact mechanism of formation of such complexes. We used the stopped‐flow and microsecond freeze‐hyperquenching (MHQ) techniques supported by steady‐state spectroscopic and electrochemical data to demonstrate the formation of partially coordinated intermediate Cu II complexes formed by glycyl‐glycyl‐histidine (GGH) peptide, the simplest ATCUN/NTS model. One of these novel intermediates, characterized by two‐nitrogen coordination, t ½ ∼100 ms at pH = 6.0 and the ability to maintain the Cu II /Cu I redox pair is the best candidate for the long‐sought reactive species in extracellular copper transport.
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ATCUN/NTS motifs participate in physiological CuII exchange. Using kinetic methods, spectroscopy, and electrochemistry, it was demonstrated that CuII binding to GGH, an ATCUN/NTS representative, proceeds via partially coordinated species. The 2N‐coordinated complex with t 1/2≈100 ms (pH 6.0) and CuII/CuI redox activity is the long‐sought reactive intermediate for extracellular copper delivery. Abstract The amino‐terminal copper and nickel/N‐terminal site (ATCUN/NTS) present in proteins and bioactive peptides exhibits high affinity towards CuII ions and have been implicated in human copper physiology. Little is known, however, about the rate and exact mechanism of formation of such complexes. We used the stopped‐flow and microsecond freeze‐hyperquenching (MHQ) techniques supported by steady‐state spectroscopic and electrochemical data to demonstrate the formation of partially coordinated intermediate CuII complexes formed by glycyl–glycyl–histidine (GGH) peptide, the simplest ATCUN/NTS model. One of these novel intermediates, characterized by two‐nitrogen coordination, t 1/2≈100 ms at pH 6.0 and the ability to maintain the CuII/CuI redox pair is the best candidate for the long‐sought reactive species in extracellular copper transport.
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Depending on the coordination, copper ions can have a very high activity in catalyzing the production of reactive oxygen species. Thus interest arose in increasing the activity of antimicrobial peptides (AMPs) by equipping them with a Cu-binding unit. Several examples, native and engineered, have been investigated with the motif Xxx-Zzz-His, called Amino Terminal Cu(II)- and Ni(II)-binding (ATCUN) motif. Here we investigate a short AMP that was equipped either with Xxx-Zzz-His or Xxx-His. Xxx-His is a shorter motif and yields a more redox active copper complex. The control AMP, Xxx-His-AMP and Xxx-Zzz-His-AMP were investigated toward Cu-binding, Reactive Oxygen Species (ROS) production and antimicrobial activity in E. coli. The data indicate that these Cu-binding motifs have very limited impact on antimicrobial activity and low ROS production capability.
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The designed “ATCUN” motif (amino-terminal copper and nickel binding site) is a replica of naturally occurring ATCUN site found in many proteins/peptides, and an attractive platform for multiple applications, which include nucleases, proteases, spectroscopic probes, imaging, and small molecule activation. ATCUN motifs are engineered at periphery by conjugation to recombinant proteins, peptides, fluorophores, or recognition domains through chemically or genetically, fulfilling the needs of various biological relevance and a wide range of practical usages. This chemistry has witnessed significant growth over the last few decades and several interesting ATCUN derivatives have been described. The redox role of the ATCUN moieties is also an important aspect to be considered. The redox potential of designed M-ATCUN derivatives is modulated by judicious choice of amino acid (including stereochemistry, charge, and position) that ultimately leads to the catalytic efficiency. In this context, a wide range of M-ATCUN derivatives have been designed purposefully for various redox- and non-redox-based applications, including spectroscopic probes, target-based catalytic metallodrugs, inhibition of amyloid-β toxicity, and telomere shortening, enzyme inactivation, biomolecules stitching or modification, next-generation antibiotic, and small molecule activation.
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We used a series of modified/substituted GGH analogues to investigate the kinetics of Cu(II) binding to ACTUN peptides. Rules for rate modulation by 1st and 2nd sphere interactions were established,...
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Preeclampsia is a blood pressure disorder associated with significant proteinuria. Hypertensive women have increased levels of neurokinin B (NKB) and Cu(II) ions in blood plasma during pregnancy. NKB bears the ATCUN/NTS N-terminal motif empowering strong Cu(II) binding in a characteristic four-nitrogen (4N) square-planar motif, but an alternative Cu(II)NKB2 geometry was proposed earlier. We studied the coordination of DMHD-NH2, representing the NKB ATCUN/NTS motif, to Cu(II) by potentiometry, electronic absorption and circular dichroism spectroscopy in water and SDS micellar solutions. NKB was studied in SDS micelles. The experiments were aided by density functional theory (DFT) calculations. We found that under all investigated conditions NKB formed solely 1 : 1 complexes. In the absence of SDS, the 4N complex at physiological pH 7.4 has a very low dissociation constant of 3.5 fM, but the interaction with SDS weakened the binding nearly thousand-fold. This interaction may serve as a molecular switch for specific Cu(II) delivery to membrane receptors by NKB. Furthermore, the calculations based on clinical data indicate a potential toxic role of Cu(II)NKB in preeclampsia.
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The complexation of Cm(III) with the recombinant human serum albumin (rHSA) (characterized by single deletion of residue Asp-1), is studied in dependence of pH and rHSA concentration using time-resolved laser fluorescence spectroscopy (TRLFS). A Cm(III) rHSA species is formed between pH 6.4 and 10.0 with the conditional stability constant being logK = 6.47 at pH = 7.4. Competition titration experiments with Cu(II) and Zn(II) confirm complexation at the N-terminal binding site (NTS) of rHSA and exclude the involvement of the Multi-Metal Binding Site (MBS). Comparison with a previous study on Cm(III) interaction with native albumin, HSA, points out, that residue Asp-1 is involved in Cm(III) binding to HSA but is not crucial for Cm(III) complexation at the NTS. The results are of major importance for a better understanding of fundamental actinide-protein interaction mechanisms which are highly required for the identification and characterization of relevant distribution pathways of incorporated radionuclides.
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ATCUN (amino terminal Cu(II) and Ni(II) binding) motifs chelate Cu(II) ions strongly. However, the impact of the phosphorylation of neighboring residues on such complexation has not been elucidated. The copper(II) dissociation constants of original and phosphorylated peptides from human histatin-1 and human serum albumin were compared using spectroscopic methods. Phosphorylation markedly weakened Cu(II) binding. Thus, these results indicate that phosphorylation may be a vital mechanism governing metal ion binding.
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The cause of Alzheimer’s disease (AD) is incompletely defined. To date, no mono-causal treatment has so far reached its primary clinical endpoints, probably due to the complexity and diverse neuropathology contributing to the neurodegenerative process. In the present paper, we describe the plausible etiological role of copper (Cu) imbalance in the disease. Cu imbalance is strongly associated with neurodegeneration in dementia, but a complete biochemical etiology consistent with the clinical, chemical, and genetic data is required to support a causative association, rather than just correlation with disease. We hypothesize that a Cu imbalance in the aging human brain evolves as a gradual shift from bound metal ion pools, associated with both loss of energy production and antioxidant function, to pools of loosely bound metal ions, involved in gain-of-function oxidative stress, a shift that may be aggravated by chemical aging. We explain how this may cause mitochondrial deficits, energy depletion of high-energy demanding neurons, and aggravated protein misfolding/oligomerization to produce different clinical consequences shaped by the severity of risk factors, additional comorbidities, and combinations with other types of pathology. Cu imbalance should be viewed and integrated with concomitant genetic risk factors, aging, metabolic abnormalities, energetic deficits, neuroinflammation, and the relation to tau, prion proteins, α-synuclein, TAR DNA binding protein-43 (TDP-43) as well as systemic comorbidity. Specifically, the Amyloid Hypothesis is strongly intertwined with Cu imbalance because amyloid-β protein precursor (AβPP)/Aβ are probable Cu/Zn binding proteins with a potential role as natural Cu/Zn buffering proteins (loss of function), and via the plausible pathogenic role of Cu-Aβ.
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Human serum albumin (HSA) and the growth factor glycyl-l-histidyl-l-lysine (GHK) bind Cu2+ as part of their normal functions. GHK is found at its highest concentration in the albumin-rich fraction of plasma, leading to speculation that HSA and GHK form a ternary Cu2+ complex. Although preliminary evidence was presented 40 years ago, the structure and stability of such a complex have remained elusive. Here, we show that two ternary Cu(GHK)NImHSA complexes are formed between GHK and the imino nitrogen (NIm) of His side chains of HSA. We identified His3 as one site of ternary complex formation (conditional binding constant cKCu(GHK)NImHis3Cu(GHK) = 2900 M–1 at pH 7.4), with the second site (cKCu(GHK)NImHisXCu(GHK) = 1700 M–1) likely being supplied by either His128 or His510. Together with the established role of HSA as a molecular shuttle in the blood, these complexes may aid the transport of the exchangeable Cu2+ pool and the functional form of GHK.
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Cu(II)-peptide complexes are intensely studied as models for biological peptides and proteins and for their direct importance in copper homeostasis and dyshomeostasis in human diseases. In particular, high-affinity ATCUN/NTS (amino-terminal copper and nickel/N-terminal site) motifs present in proteins and peptides are considered as Cu(II) transport agents for copper delivery to cells. The information on the affinities and structures of such complexes derived from steady-state methods appears to be insufficient to resolve the mechanisms of copper trafficking, while kinetic studies have recently shown promise in explaining them. Stopped-flow experiments of Cu(II) complexation to ATCUN/NTS peptides revealed the presence of reaction steps with rates much slower than the diffusion limit due to the formation of novel intermediate species. Herein, the state of the field in Cu(II)-peptide kinetics is reviewed in the context of physiological data, leading to novel ideas in copper biology, together with the discussion of current methodological issues.
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Two amide group containing pyridine derivatives, N-(pyridin-2-ylmethyl)picolinamide (PMPA) and N-(pyridin-2-ylmethyl)-2-((pyridin-2-ylmethyl)amino)acetamide (DPMGA), have been investigated as potential metallophores in the therapy of Alzheimer's disease. Their complex formation with Cu(II) and Zn(II) were characterized in details. Unexpectedly not only the Cu(II) but also the Zn(II) was able to induce deprotonation of the amide-NH, however, it occurred only at higher pH or at higher metal ion concentrations than the biological conditions. At μM concentration level mono complexes (MLH−1) dominate with both ligands. Direct fluorescence and reactive oxygen species (ROS) producing measurements prove that both ligands are able to remove Cu(II) from its amyloid-β complexes (CuAβ). Correlation was also established between the conditional stability constant of the Cu(II) complexes with different ligands and their ability of inhibition of ROS production by CuAβ.
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Ctr1 regulates copper uptake and its intracellular distribution. The first 14 amino acid sequence of the Ctr1 ectodomain Ctr1(1-14) encompasses the characteristic Amino Terminal Cu2+ and Ni2+ binding motif (ATCUN) as well as the bis-His binding motif (His5 and His6). We report a combined thermodynamic and spectroscopic (UV-vis, CD, EPR) study dealing with the formation of Cu2+ homobinuclear complexes with Ctr1(1-14), the percentage of which is not negligible even in the presence of a small Cu2+ excess and clearly prevails at a M/L ratio of 1.9. Ascorbate fails to reduce Cu2+ when bound to the ATCUN motif, while it reduces Cu2+ when bound to the His5-His6 motif involved in the formation of binuclear species. The histidine diade characterizes the second binding site and is thought to be responsible for ascorbate oxidation. Binding constants and speciation of Ag+ complexes with Ctr1(1-14), which are assumed to mimic Cu+ interaction with N-terminus of Ctr1(1-14), were also determined. A preliminary immunoblot assay evidences that the anti-Ctr1 extracellular antibody recognizes Ctr1(1-14) in a different way from the longer Ctr1(1-25) that encompasses a second His and Met rich domain.
Article
The amyloid-β (Aβ) peptide is a cleavage product of the amyloid precursor protein and has been implicated as a central player in Alzheimer's disease. The N-terminal end of Aβ is variable, and different proportions of these variable-length Aβ peptides are present in healthy individuals and those with the disease. The N-terminally truncated form of Aβ starting at position 4 (Aβ4-x) has a His residue as the third amino acid (His6 using the formal Aβ numbering). The N-terminal sequence Xaa-Xaa-His is known as an amino terminal copper and nickel binding motif (ATCUN), which avidly binds Cu(II). This motif is not present in the commonly studied Aβ1-x peptides. In addition to the ATCUN site, Aβ4-x contains an additional metal binding site located at the tandem His residues (bis-His at His13 and 14) which is also found in other isoforms of Aβ. Using the ATCUN and bis-His motifs, the Aβ4-x peptide is capable of binding multiple metal ions simultaneously. We confirm that Cu(II) bound to this particular ATCUN site is redox silent, but the second Cu(II) site is redox active and can be readily reduced with ascorbate. We have employed surrogate metal ions to block copper coordination at the ATCUN or the tandem His site in order to isolate spectral features of the copper coordination environment for structural characterization using extended X-ray absorption fine structure (EXAFS) spectroscopy. This approach reveals that each copper coordination environment is independent in the Cu2Aβ4-x state. The identification of two functionally different copper binding environments within the Aβ4-x sequence may have important implications for this peptide in vivo.
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Peptides and proteins with N-terminal amino acid sequences NH2-Xxx-His (XH) and NH2-Xxx-Zzz-His (XZH) form well established high affinity CuII-complexes. Key examples are Asp-Ala-His (in serum albumin) and Gly-His-Lys, the wound healing factor. This opens a straightforward way to add a high affinity CuII-binding site to almost any peptide or protein, by chemical or recombinant approaches. Thus, these motifs, NH2-Xxx-Zzz-His in particular, have been used to equip peptides and proteins with a multitude of functions based on the redox activity of Cu, including nuclease, protease, glycosidase, or oxygen activation, useful in anticancer or antimicrobial drugs. More recent research suggest novel biological function, mainly based on the redox inertness of CuII in XZH, like PET imaging (with 64Cu), chelation therapies (for instance in Alzheimer's disease and other types of neurodegeneration), antioxidant units, Cu transporters and activation of biological functions by strong CuII binding. This review gives an overview on the chemical properties of Cu-XH and -XZH motifs and discusses the pros and cons for the vast and different biological applications, and how they could be improved depending on the application.
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Human serum albumin (HSA)-based drug delivery systems are promising for improving delivery efficiency, anticancer activity and selectivity of anticancer agents. To rationally guide to design HSA carrier for anticancer metal agent, we built a breast mouse model on developing anti-cancer copper (Cu) pro-drug based on the nature of IIA subdomain of HSA carrier and cancer cells. Thus, we first synthesized a new Cu(II) compound derived from tridentate (E)-N'-(5-bromo-2-hydroxybenzylidene)benzohydrazide Schiff base ligand (HL) containing 2 potential leaving groups [indazole (Ind) and NO3-], namely, [Cu(L)(Ind)NO3]. Structural analysis of the HSA complex showed that Cu(L)(Ind)(NO3) could bind to the hydrophobic pocket of the HSA IIA subdomain. Lys199 and His242 coordinate with Cu2+ by replacing the indazole and NO3 ligands of [Cu(L)(Ind)NO3]. The release behavior of the Cu compound from the HSA complex is different at different pH levels. [Cu(L)(Ind)NO3] can enhance cytotoxicity by 2 times together with HSA specifically in cancer cells but has no such effect on normal cells in vitro. Importantly, our in vivo results showed that the HSA complex displayed increased selectivity and capacity to inhibit tumor growth and was less toxic than [Cu(L)(Ind)NO3] alone.
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Physicochemical studies of the human serum albumin-Cu(II)-l-histidine (HSA-Cu(II)-l-His) ternary complex at pH 7.5 indicate several interesting features. 1. The dissociation constants of the HSA-Cu(II)-l-His ternary complex and the HSA-Cu(II) binary complex are 1.38 x 10⁻²² and 6.61 x 10⁻¹⁷, respectively. 2. The absorption spectrum of the ternary complex system has characteristics similar to those of the NH2-terminal peptide (1-24) of bovine serum albumin in the presence of Cu(II) and l-His. The computed spectrum of HSA-Cu(II)-l-His has a λmax at 540 nm, a shift of 15 nm toward red from that of HSA-Cu(II) (λmax = 525 nm). 3. The calculated moles of protons liberated in the formation of HSA-Cu(II)-l-His from HSA and Cu(II)-l-His2 are 0.28. While this may originate from an α-amino group, the number is much less than what would be expected if a peptide nitrogen of HSA were involved in the binding. 4. Cu(II) in the ternary complex is most likely bound to both HSA and l-His. The possible binding mode changes from all nitrogen, as shown in HSA-Cu(II), to either a mixture of nitrogen and oxygen or an additional involvement of imidazole nitrogen. 5. Equilibria, existing in the ternary complex system, suggest that the ternary complex may play an important role in the biological transport of Cu(II).
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Intrinsic stoichiometric equilibrium constants were determined for zinc(II) and copper(II) binding to bovine and human serum albumin. Data were obtained from equilibrium dialysis experiments. Metals were presented to apoprotein as metal chelates in order to avoid metal hydrolysis and to minimize nonspecific metal-protein interactions. Scatchard analysis of the binding data indicated that the high affinity class for both zinc and copper was comprised of one site. Results of binding experiments done at several pH values suggested that while both histidyl and carboxyl groups appear to be involved in copper binding, histidyl residues alone were sufficient for zinc binding. These amino acid residues were used in combination to model several binding sites used in the formulation of equilibria expressions from which stoichiometric constants were calculated. The log10K for bovine serum albumin were calculated to be 7.28 for Zn(II) and 11.12 for Cu(II). Those for human serum albumin were determined to be 7.53 and 11.18 for Zn(II) and Cu(II), respectively. These constants were used in equilibria to simulate speciation of metal-albumin and metal-chelator and to illustrate relative binding affinities. This comparison of binding strengths was possible only through the calculation of an intrinsic stoichiometric binding constant.
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The copper(II) complex of 3,5-diisopropylsalicylate is a lipophilic water-insoluble binuclear complex, Cu(II)2(3,5-DIPS)4, that has attracted interest because of a wide range of pharmacological activities. This study was undertaken to examine bonding interactions between the complex and human serum albumin (HSA) to help elucidate the mode of transport of the complex in vivo. Electron paramagnetic resonance, numerical magnetic resonance and UV-visible absorption spectroscopic studies were performed using 200 microM aqueous solutions (pH 7.5) of HSA to which had been added up to three molar equivalents of CuCl2, CuSO4, or Cu(II)2(3,5-DIPS)4. Both EPR and UV-visible spectra demonstrated the presence of more than one copper bonding site on HSA, and proton NMR spectra showed that the 3,5-DIPS ligand is also bonded to HSA. These results indicate that there is no observable direct coordination of the ligand to copper in the presence of HSA, and that the majority of the copper and 3,5-DIPS bond to HSA at separate sites. Addition of solid Cu(II)2(3,5-DIPS)4 to HSA at pH 7.5 similarly resulted in spectra suggest that there are no ternary Cu(II)(3,5-DIPS), Cu(II)(3,5-DIPS)2, or Cu(II)2(3,5-DIPS)4 complexes formed with HSA. It is concluded that any ternary complexes formed in the presence of HSA are below the spectroscopic detection limits and represent less than 5% of the total copper.
Article
Human cells acquire copper primarily via the copper transporter 1 protein, hCtr1. We demonstrate that at extracellular pH 7.4 Cu(II) is bound to the model peptide hCtr1_1-14 via an ATCUN motif and such complexes are strong enough to collect Cu(II) from albumin, supporting the potential physiological role of Cu(II) binding to hCtr1.
Article
Copper Transporter 1 (CTR1) is a homotrimeric membrane protein providing the main route of copper transport into eukaryotic cells from the extracellular milieu. Its N-terminal extracellular domain, rich in His and Met residues, is considered responsible for directing copper into the transmembrane channel. Most of vertebrate CTR1 proteins contain the His residue in position three from N-terminus, creating a well-known Amino Terminal Cu(II)- and Ni(II)-Binding (ATCUN) site. CTR1 from humans, primates and many other species contains the Met-Asp-His (MDH) sequence, while some rodents including mouse have the Met-Asn-His (MNH) N-terminal sequence. CTR1 is thought to collect Cu(II) ions from blood copper transport proteins, including albumin, but previous reports indicated that the affinity of N-terminal peptide/domain of CTR1 is significantly lower than that of albumin, casting serious doubt on this aspect of CTR1 function. Using potentiometry and spectroscopic techniques we demonstrated that MDH-amide, a tripeptide model of human CTR1 N-terminus, binds Cu(II) with K of 1.3 × 1013 M-1 at pH 7.4, ~13 times stronger than Human Serum Albumin (HSA), and MNH-amide is even stronger, K of 3.2 × 1014 M-1 at pH 7.4. These results indicate that the N-terminus of CTR1 may serve as intermediate binding site during Cu(II) transfer from blood copper carriers to the transporter. MDH-amide, but not MNH-amide also forms a low abundance complex with non-ATCUN coordination involving the Met amine, His imidazole and Asp carboxylate. This species might assist Cu(II) relay down the peptide chain or its reduction to Cu(I), both steps necessary for the CTR1 function.
Article
Human serum albumin (HSA) is a major Cu carrier in human blood and in cerebrospinal fluid. A major assumption is that Cu bound to HSA is in the Cu(II) oxidation state; thus, interactions between HSA and Cu(II) have been intensely investigated for over four decades. HSA has been reported previously to support the reduction of Cu(II) to the Cu(I) oxidation state in the presence of the weak reductant, ascorbate; however, the interactions between HSA and Cu(I) have not been explicitly investigated. Here, we characterize both the apparent affinity of HSA for Cu(I) using solution competition experiments and the coordination structure of Cu(I) bound to HSA using X-ray absorption spectroscopy and in silico modeling. We find that HSA binds to Cu(I) at pH 7.4 with an apparent conditional affinity of KCu(I):HSA = 10(14.0) using digonal coordination in a structure that is similar to the bis-His coordination modes characterized for amyloid beta (Aβ) and the prion protein. This high affinity and familiar Cu(I) coordination structure suggests that Cu(I) interaction with HSA in human extracellular fluids is unappreciated in the current scientific literature.
Article
We know that blood plasma contains many proteins and also other components that bind copper. The largest contributor to copper in the plasma is ceruloplasmin, which accounts for 40-70 percent. Apart from ceruloplasmin and albumin, most of these components have not been studied extensively, and even for ceruloplasmin and albumin, much remains to be discovered. New components with new functions, and new functions of known components are emerging, some warranting reconsideration of earlier findings. The author's laboratory has been actively involved in research on this topic. This review summarizes and updates our knowledge of the nature and functions of ceruloplasmin and the other known and emerging copper-containing molecules (principally proteins) in this fluid, to better understand how they contribute to copper homeostasis and consider their potential significance to health and disease.
Article
Accumulation of the β-amyloid (Aβ) peptide in extracellular senile plaques rich in copper and zinc is a defining pathological feature of Alzheimer's disease (AD). The Aβ1-x (x=16/28/40/42) peptides have been the primary focus of Cu(II) binding studies for more than 15 years; however, the N-truncated Aβ4-42 peptide is a major Aβ isoform detected in both healthy and diseased brains, and it contains a novel N-terminal FRH sequence. Proteins with His at the third position are known to bind Cu(II) avidly, with conditional log K values at pH 7.4 in the range of 11.0-14.6, which is much higher than that determined for Aβ1-x peptides. By using Aβ4-16 as a model, it was demonstrated that its FRH sequence stoichiometrically binds Cu(II) with a conditional Kd value of 3×10(-14) M at pH 7.4, and that both Aβ4-16 and Aβ4-42 possess negligible redox activity. Combined with the predominance of Aβ4-42 in the brain, our results suggest a physiological role for this isoform in metal homeostasis within the central nervous system. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Article
ESR is useful for determining the unpaired electronic state around paramagnetic metal ions such as, copper(II), iron(III) and manganese(II). The coordination structure of Cu(II) complexes was examined by ESR. ESR spectra were measured for a series of polyamine Cu(II) complexes, whose coordination modes are square-planar, axially-coordinate square-planar, tetrahedral and distorted square-planar (copper ion displaced from N4 plane). ESR parameters, g-values and hyperfine coupling constants, A-values, were found to be correlated with the coordination mode of the complex, in which g vs. A plots gave important information on coordination structure of the complex. Moreover, linear relationships between ESR parameters and stability constants or halfwave potentials of the Cu(II) complexes were observed. Based on these results, the coordination structure around the Cu(II) in copper complexes as well as copper proteins was predicted by ESR spectra and associated parameters.
Article
The review describes the state of the art in the field of stability constant determination for Cu(II), Cu(I) and Zn(II) complexes of proteins and peptides involved in neurodegenerative diseases, α-synuclein (aS), prion protein (PrP), amyloid precursor protein (APP) and amyloid β peptides (Aβ). The methodologies and results are critically analyzed and recommendations are formulated about possible systematic errors in these studies. The possibility of formation of ternary complexes with titration competitors is discussed.
Article
Thermodynamic and spectroscopic studies have shown that the insertion of alpha-hydroxylmethylserine (HmS) residues into the N-terminal peptide motif of human serum albumin results in a very powerful chelating agent for Cu2+ and Ni2+ ions. The insertion of two HmS residues results in the HmS-HmS-His-OH/NH2 sequence, which is the most effective chelating agent based on an albumin-like sequence for both studied metal ions, especially when the C-terminal carboxylate is protected by an amide function.
Article
The synthesis of the tetrapeptides Ala-Gly-Gly-His, Boc-Ala-Gly-Gly-His (Boc = t-butoxycarbonyl), Ala-Gly-Gly-His(π-bom)(π-bom =Nπ-benzoxymethyl), Ala-Gly-Gly-His-OMe, and Ala-Gly-Pro-His is reported, together with the results of a pH-metric and spectroscopic (absorption, c.d., and e.s.r.) study of their complexes with H+ and CuII. The work was designed to study the initial site of binding to CuII in peptides containing both a terminal amino nitrogen and a histidyl residue. Results show that the π-N of the imidazole ring of the histidyl residue is the primary anchoring site for copper(II) co-ordination, and that the next nitrogen to bond can be the terminal amino N, forming a macrocyclic chelate ring.
Article
Serum albumin is the most abundant protein in the blood and cerebrospinal fluid and plays a fundamental role in the distribution of essential transition metal ions in the human body. Human serum albumin (HSA) is an important physiological transporter of the essential metal ions Cu(2+), and Zn(2+) in the bloodstream. Its binding of metals like Ni(2+), Co(2+), or Cd(2+) can occur in vivo, but is only of toxicological relevance. Moreover, HSA is one of the main targets and hence most studied binding protein for metallodrugs based on complexes with Au, Pt and V. We discuss i) the four metal-binding sites so far described on HSA, their localization and metal preference, ii) the binding of the metal ions mentioned above, i.e. their stability constants and association/dissociation rates, their coordination chemistry and their selectivity versus the four binding sites iii) the methodology applied to study issues of items i and ii and iv) oligopeptide models of the N-terminal binding site. Albumin has four partially selective metal binding sites with well-defined metal preferences. It is an important regulator of the blood transport of physiological Cu(II) and Zn(II) and toxic Ni(II) and Cd(II). It is also an important target for metal-based drugs containing Pt(II), V(IV)O, and Au(I). The thorough understanding of metal binding properties of serum albumin, including the competition of various metal ions for specific binding sites is important for biomedical issues, such as new disease markers and design of metal-based drugs. This article is part of a Special Issue entitled Serum Albumin.
Article
The complexation of Cu(I) and Cu(II) by a series of 12-, 14- and 16-membered macrocyclic ligands 1–6 containing the N2S2 donor set has been studied potentiometrically, spectrophotometrically and voltammetrically. In the case of Cu(II), mononuclear complexes CuL2+ with stability constants of 1010–1015 are formed. In addition, partially hydrolyzed species Cu(L)OH+ are observed at pH > 10 for the 12-membered ligands. For Cu(I), beside the specis CuL+ with stabilities of 1012–1014, the unexpected formation of protonated species CuLH2+ was detected. In contrast to the well-known general trends in coordination chemistry, the stability of these protonated species increases relative to that of the complexes with the neutral ligand when the ring size and concomitantly the distance between neighbouring donor atoms is decreased. From the stability constants of the Cu(I)- and Cu(II)-complexes the redox potentials have been calculated and are compared to the values of E1/2 obtained by cyclic voltammetry. Despite the identical donor set the Cu(II)/Cu(I) redox potentials of the complexes are spanning a range of 340 mV or six orders of magnitude in relative stability, reflecting the importance of subtle differences in steric requirements.
Article
Circular dichroism and electron spin resonance spectroscopy are used to investigate the second specific metal binding site on human, bovine and porcine albumins. Ni(II), Zn(II) and Cd(II) can displace Cu(II) from the second Cu(II) site but not from the first strong site of human and bovine albumins (the N-terminal site). The second Cu(II) binds more strongly than the other metal ions to the second site of all three proteins, except Zn(II) binding to porcine albumin which is ca. 10 × stronger than Cu(II). The second Cu(II) site appears to be a tetragonal {2N,4O} site.
Article
Human serum albumin (HSA), the most abundant protein in plasma, is a monomeric multi-domain macromolecule, representing the main determinant of plasma oncotic pressure and the main modulator of fluid distribution between body compartments. HSA displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds. Indeed, HSA represents the main carrier for fatty acids, affects pharmacokinetics of many drugs, provides the metabolic modification of some ligands, renders potential toxins harmless, accounts for most of the anti-oxidant capacity of human plasma, and displays (pseudo-)enzymatic properties. HSA is a valuable biomarker of many diseases, including cancer, rheumatoid arthritis, ischemia, post-menopausal obesity, severe acute graft-versus-host disease, and diseases that need monitoring of the glycemic control. Moreover, HSA is widely used clinically to treat several diseases, including hypovolemia, shock, burns, surgical blood loss, trauma, hemorrhage, cardiopulmonary bypass, acute respiratory distress syndrome, hemodialysis, acute liver failure, chronic liver disease, nutrition support, resuscitation, and hypoalbuminemia. Recently, biotechnological applications of HSA, including implantable biomaterials, surgical adhesives and sealants, biochromatography, ligand trapping, and fusion proteins, have been reported. Here, genetic, biochemical, biomedical, and biotechnological aspects of HSA are reviewed.
Article
Cellular acquisition of copper in eukaryotes is primarily accomplished through the Ctr family of copper transport proteins. In both humans and yeast, methionine-rich "Mets" motifs in the amino-terminal extracellular domain of Ctr1 are thought to be responsible for recruitment of copper at the cell surface. Unlike yeast, mammalian Ctr1 also contains extracellular histidine-rich motifs, although a role for these regions in copper uptake has not been explored in detail. Herein, synthetic model peptides containing the first 14 residues of the extracellular domain of human Ctr1 (MDHSHHMGMSYMDS) have been prepared and evaluated for their apparent binding affinity to both Cu(I) and Cu(II). These studies reveal a high affinity Cu(II) binding site (log K = 11.0 ± 0.3 at pH 7.4) at the amino-terminus of the peptide as well as a high affinity Cu(I) site (log K = 10.2 ± 0.2 at pH 7.4) that utilizes adjacent HH residues along with an additional His or Met ligand. These model studies suggest that the histidine domains may play a direct role in copper acquisition from serum copper-binding proteins and in facilitating the reduction of Cu(II) to the active Ctr1 substrate, Cu(I). We tested this hypothesis by expressing a Ctr1 mutant lacking only extracellular histidine residues in Ctr1-knockout mouse embryonic fibroblasts. Results from live cell studies support the hypothesis that extracellular amino-terminal His residues directly participate in the copper transport function of Ctr1.
Article
The protonation constants of the tripeptide glycylglycyl-L-histidine (L-) have been determined at 25 degrees C and I = 0.1 mol dm-3 as log K 8.06, 6.82, and 2.80. Complexation with copper(II) can be represented by the series of equilibria [formula: see text] in the case of nickel(II) only the species [NiLH]2+, [NiL]+, and [NiLH-2]- are of importance with log beta 111 = 11.33(2); log beta 110 = 4.74(6), and log beta 11-2 = -6.93(1). The tripeptide acts as a quadridentate ligand to give complexes with copper and nickel with an amino group, two deprotonated amide groups and an imidazole pyridine nitrogen (Im-N3) as donors. At 1:1 ligand-to-metal ratios the purple copper(II) complex [CuLH-2]- is essentially 100% abundant above pH 7 and the planar yellow [NiLH-2]- above pH 8. The displacement of the tripeptide ligand from the nickel(II) complex by L-histidine has been studied kinetically over the pH range 7-8. There is a small solvolytic reaction and a reaction which is first-order in the hydrogen ion concentration. Under the experimental conditions employed, the reaction is essentially independent of the L-His concentration and displacement occurs by a proton-assisted nucleophilic pathway with rate-determining cleavage of the first nickel(II)-N(peptide) bond.
Article
A comparative study of thermodynamic and kinetic aspects of Cu(II) and Ni(II) binding at the N-terminal binding site of human and bovine serum albumins (HSA and BSA, respectively) and short peptide analogues was performed using potentiometry and spectroscopic techniques. It was found that while qualitative aspects of interaction (spectra and structures of complexes, order of reactions) could be reproduced, the quantitative parameters (stability and rate constants) could not. The N-terminal site in HSA is much more similar to BSA than to short peptides reproducing the HSA sequence. A very strong influence of phosphate ions on the kinetics of Ni(II) interaction was found. This study demonstrates the limitations of short peptide modelling of Cu(II) and Ni(II) transport by albumins.
Article
Serum albumin can specifically bind one Cu(II)-ion, and is proposed to function as a copper transport protein in vivo. Cu(II)-albumin is rapidly reduced by ascorbate. A second order rate constant of 0.54 mM(-l) x min(-1) was estimated for the reaction. The oxidation process is catalytic, the Cu(I)-albumin molecule being reoxidized by molecular oxygen. The reaction was found to follow Michaelis-Menten kinetics, characterized by an apparent Km-value of 0.89 mM, and a catalytic constant of 0.066 microM O2/min. An apparent inhibition of oxygen uptake was obtained with catalase (but not with superoxide dismutase), suggesting the formation of H2O2 in the system. Wilson's disease patients usually have increased amounts of non-ceruloplasmin copper in plasma. The low level of plasma ascorbate observed in such patients could possibly be due, at least in part, to an oxidation by Cu(II)-albumin.
Article
GSH and L-His are abundant biomolecules and likely biological ligands for Zn(II) under certain conditions. Potentiometric titrations provide evidence of formation of ternary Zn(II) complexes with GSH and L-His or D-His with slight stereoselectivity in favour of L-His (ca. 1 log unit of stability constant). The solution structure of the ZnH(GSH)(L-His)(H2O) complex at pH 6.8, determined by NMR, includes tridentate L-His, monodentate (sulfur) GSH, and weak interligand interactions. Calculations of competitivity of this complex for Zn(II) binding at pH 7.4 indicate that it is likely to be formed in vivo under conditions of GSH depletion. Otherwise, GSH alone emerges as a likely Zn(II) carrier.
Article
The conditional stability constant at pH 7.4 for Cu(II) binding at the N-terminal site (NTS) of human serum albumin (HSA) was determined directly by competitive UV–vis spectroscopy titrations using nitrilotriacetic acid (NTA) as the competitor in 100 mM NaCl and 100 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (Hepes). The log K NTSc value of 12.0 ± 0.1 was determined for HSA dissolved in 100 mM NaCl. A false log log K NTSc value of 11.4 ± 0.1 was obtained in the 100 mM Hepes buffer, owing to the formation of a ternary Cu(NTA)(Hepes) complex. The impact of the picomolar affinity of HSA for Cu(II) on the availability of these ions in neurodegenerative disorders is briefly discussed.
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M. Mital, N. E. Wezynfeld, T. Frączyk, M. Z. Wiloch, U. E. Wawrzyniak, A. Bonna, C. Tumpach, K. J. Barnham, C. L. Haigh, W. Bal, S. C. Drew, Angew. Chem. Int. Ed. 2015, 54, 10460-10464.
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