α-lipoic acid ameliorates consequences of copper overload by up-regulating selenoproteins and decreasing redox misbalance.

α-lipoic acid (LA) is an essential cofactor for mitochondrial dehydrogenases and is required for cell growth, metabolic fuel production, and antioxidant defense. In vitro, LA binds copper (Cu) with high affinity and as an endogenous membrane permeable metabolite could be advantageous in mitigating the consequences of Cu overload in human diseases. We tested this hypothesis in 3T3-L1 preadipocytes with inactivated Cu transporter Atp7a; these cells accumulate Cu and show morphologic changes and mitochondria impairment. Treatment with LA corrected the morphology of Atp7a-/- cells similar to the Cu chelator bathocuproinedisulfonate (BCS) and improved mitochondria function; however, the mechanisms of LA and BCS action were different. Unlike BCS, LA did not decrease intracellular Cu but instead increased selenium levels that were low in Atp7a-/- cells. Proteome analysis confirmed distinct cell responses to these compounds and identified upregulation of selenoproteins as the major effect of LA on preadipocytes. Upregulation of selenoproteins was associated with an improved GSH:GSSG ratio in cellular compartments, which was lowered by elevated Cu, and reversal of protein oxidation. Thus, LA diminishes toxic effects of elevated Cu by improving cellular redox environment. We also show that selenium levels are decreased in tissues of a Wilson disease animal model, especially in the liver, making LA an attractive candidate for supplemental treatment of this disease.

Evaluation of Zn2+- and Cu2+-Binding Affinities of Native Cu,Zn-SOD1 and Its G93A Mutant by LC-ICP MS.

The tight binding of Cu and Zn ions to superoxide dismutase 1 (SOD1) maintains the protein stability, associated with amyotrophic lateral sclerosis (ALS). Yet, the quantitative studies remain to be explored for the metal-binding affinity of wild-type SOD1 and its mutants. We have investigated the demetallation of Cu,Zn-SOD1 and its ALS-related G93A mutant in the presence of different standard metal ion chelators at varying temperatures by using an LC-ICP MS-based approach and fast size-exclusion chromatography. Our results showed that from the slow first-order kinetics both metal ions Zn2+ and Cu2+ were released simultaneously from the protein at elevated temperatures. The rate of the release depends on the concentration of chelating ligands but is almost independent of their metal-binding affinities. Similar studies with the G93A mutant of Cu,Zn-SOD1 revealed slightly faster metal-release. The demetallation of Cu,Zn-SOD1 comes always to completion, which hindered the calculation of the KD values. From the Arrhenius plots of the demetallation in the absence of chelators ΔH‡ = 173 kJ/mol for wt and 191 kJ/mol for G93A mutant Cu,Zn-SOD1 was estimated. Obtained high ΔH values are indicative of the occurrence of protein conformational changes before demetallation and we concluded that Cu,Zn-SOD1 complex is in native conditions kinetically inert. The fibrillization of both forms of SOD1 was similar.

Affinity gradients drive copper to cellular destinations.

Copper is an essential trace element for eukaryotes and most prokaryotes. However, intracellular free copper must be strictly limited because of its toxic side effects. Complex systems for copper trafficking evolved to satisfy cellular requirements while minimizing toxicity. The factors driving the copper transfer between protein partners along cellular copper routes are, however, not fully rationalized. Until now, inconsistent, scattered and incomparable data on the copper-binding affinities of copper proteins have been reported. Here we determine, through a unified electrospray ionization mass spectrometry (ESI-MS)-based strategy, in an environment that mimics the cellular redox milieu, the apparent Cu(I)-binding affinities for a representative set of intracellular copper proteins involved in enzymatic redox catalysis, in copper trafficking to and within various cellular compartments, and in copper storage. The resulting thermodynamic data show that copper is drawn to the enzymes that require it by passing from one copper protein site to another, exploiting gradients of increasing copper-binding affinity. This result complements the finding that fast copper-transfer pathways require metal-mediated protein-protein interactions and therefore protein-protein specific recognition. Together with Cu,Zn-SOD1, metallothioneins have the highest affinity for copper(I), and may play special roles in the regulation of cellular copper distribution; however, for kinetic reasons they cannot demetallate copper enzymes. Our study provides the thermodynamic basis for the kinetic processes that lead to the distribution of cellular copper.

Metallothionein 2A affects the cell respiration by suppressing the expression of mitochondrial protein cytochrome c oxidase subunit II.

Metallothioneins (MT) are involved in a broad range of cellular processes and play a major role in protection of cells towards various stressors. Two functions of MTs, namely the maintaining of the homeostasis of transition metal ions and the redox balance, are directly linked to the functioning of mitochondria. Dyshomeostasis of MTs is often related with malfunctioning of mitochondria; however, the mechanism by which MTs affect the mitochondrial respiratory chain is still unknown. We demonstrated that overexpression of MT-2A in HEK cell line decreased the oxidative phosphorylation capacity of the cells. HEK cells overexpressing MT-2A demonstrated reduced oxygen consumption and lower cellular ATP levels. MT-2A did not affect the number of mitochondria, but reduced specifically the level of cytochrome c oxidase subunit II protein, which resulted in lower activity of the complex IV.

Human superoxide dismutase 1 (hSOD1) maturation through interaction with human copper chaperone for SOD1 (hCCS).

Copper chaperone for superoxide dismutase 1 (SOD1), CCS, is the physiological partner for the complex mechanism of SOD1 maturation. We report an in vitro model for human CCS-dependent SOD1 maturation based on the study of the interactions of human SOD1 (hSOD1) with full-length WT human CCS (hCCS), as well as with hCCS mutants and various truncated constructs comprising one or two of the protein's three domains. The synergy between electrospray ionization mass spectrometry (ESI-MS) and NMR is fully exploited. This is an in vitro study of this process at the molecular level. Domain 1 of hCCS is necessary to load hSOD1 with Cu(I), requiring the heterodimeric complex formation with hSOD1 fostered by the interaction with domain 2. Domain 3 is responsible for the catalytic formation of the hSOD1 Cys-57-Cys-146 disulfide bond, which involves both hCCS Cys-244 and Cys-246 via disulfide transfer.

Formation of [4Fe-4S] clusters in the mitochondrial iron-sulfur cluster assembly machinery.

The generation of [4Fe-4S] clusters in mitochondria critically depends, in both yeast and human cells, on two A-type ISC proteins (in mammals named ISCA1 and ISCA2), which perform a nonredundant functional role forming in vivo a heterocomplex. The molecular function of ISCA1 and ISCA2 proteins, i.e., how these proteins help in generating [4Fe-4S] clusters, is still unknown. In this work we have structurally characterized the Fe/S cluster binding properties of human ISCA2 and investigated in vitro whether and how a [4Fe-4S] cluster is assembled when human ISCA1 and ISCA2 interact with the physiological [2Fe-2S](2+) cluster-donor human GRX5. We found that (i) ISCA2 binds either [2Fe-2S] or [4Fe-4S] cluster in a dimeric state, and (ii) two molecules of [2Fe-2S](2+) GRX5 donate their cluster to a heterodimeric ISCA1/ISCA2 complex. This complex acts as an "assembler" of [4Fe-4S] clusters; i.e., the two GRX5-donated [2Fe-2S](2+) clusters generate a [4Fe-4S](2+) cluster. The formation of the same [4Fe-4S](2+) cluster-bound heterodimeric species is also observed by having first one [2Fe-2S](2+) cluster transferred from GRX5 to each individual ISCA1 and ISCA2 proteins to form [2Fe-2S](2+) ISCA2 and [2Fe-2S](2+) ISCA1, and then mixing them together. These findings imply that such heterodimeric complex is the functional unit in mitochondria receiving [2Fe-2S] clusters from hGRX5 and assembling [4Fe-4S] clusters before their transfer to the final target apo proteins.

Effect of agitation on the peptide fibrillization: Alzheimer's amyloid-ß peptide 1-42 but not amylin and insulin fibrils can grow under quiescent conditions.

Many peptides and proteins can form fibrillar aggregates in vitro, but only a limited number of them are forming pathological amyloid structures in vivo. We studied the fibrillization of four peptides--Alzheimer's amyloid-ß (Aß) 1-40 and 1-42, amylin and insulin. In all cases, intensive mechanical agitation of the solution initiated fast fibrillization. However, when the mixing was stopped during the fibril growth phase, the fibrillization of amylin and insulin was practically stopped, and the rate for Aß40 substantially decreased, whereas the fibrillization of Aß42 peptide continued to proceed with almost the same rate as in the agitated conditions. The reason for the different sensitivity of the in vitro fibrillization of these peptides towards agitation in the fibril growth phase remains elusive.

The role of initial oligomers in amyloid fibril formation by human stefin B.

Oligomers are commonly observed intermediates at the initial stages of amyloid fibril formation. They are toxic to neurons and cause decrease in neural transmission and long-term potentiation. We describe an in vitro study of the initial steps in amyloid fibril formation by human stefin B, which proved to be a good model system. Due to relative stability of the initial oligomers of stefin B, electrospray ionization mass spectrometry (ESI MS) could be applied in addition to size exclusion chromatography (SEC). These two techniques enabled us to separate and detect distinguished oligomers from the monomers: dimers, trimers, tetramers, up to decamers. The amyloid fibril formation process was followed at different pH and temperatures, including such conditions where the process was slow enough to detect the initial oligomeric species at the very beginning of the lag phase and those at the end of the lag phase. Taking into account the results of the lower-order oligomers transformations early in the process, we were able to propose an improved model for the stefin B fibril formation.

Direct Competition of ATCUN Peptides with Human Serum Albumin for Copper(II) Ions Determined by LC-ICP MS.

Copper is an indispensable biometal, primarily serving as a redox-competent cofactor in numerous proteins. Apart from preformed copper-binding sites within the protein structures, small peptide motifs exist called ATCUN, which are composed of an N-terminal tripeptide XZH, able to bind Cu(II) ions in exchangeable form. These motifs are common for serum albumin, but they are also present in a wide range of proteins and peptides. These proteins and peptides can be involved in copper metabolism, and copper ions can affect their biological role. The distribution of copper between the ATCUN peptides, including truncated amyloid-ß (Aß) peptides Aß4-42 and Aß11-42, which may be involved in Alzheimer's disease pathogenesis, is mainly determined by their concentrations and relative Cu(II)-binding affinities. The Cu(II)-binding affinity (log Kd) of several ATCUN peptides, determined by different methods and authors, varies by more than three orders of magnitude. This variation may be attributed to the chemical properties of peptides but can also be influenced by the differences in methods and experimental conditions used for the determination of Kd. In the current study, we performed direct competition experiments between selected ATCUN peptides and HSA by using an LC-ICP MS-based approach. We demonstrated that ATCUN and truncated Aß peptides Aß4-16 and Aß11-15 bind Cu(II) ions with an affinity similar to that for HSA. Our results demonstrate that ATCUN motifs cannot compete with excess HSA for the binding of Cu(II) ions in the blood and cerebrospinal fluid.

Characterization of Uranyl (UO22+) Ion Binding to Amyloid Beta (Aß) Peptides: Effects on Aß Structure and Aggregation.

Uranium (U) is naturally present in ambient air, water, and soil, and depleted uranium (DU) is released into the environment via industrial and military activities. While the radiological damage from U is rather well understood, less is known about the chemical damage mechanisms, which dominate in DU. Heavy metal exposure is associated with numerous health conditions, including Alzheimer's disease (AD), the most prevalent age-related cause of dementia. The pathological hallmark of AD is the deposition of amyloid plaques, consisting mainly of amyloid-ß (Aß) peptides aggregated into amyloid fibrils in the brain. However, the toxic species in AD are likely oligomeric Aß aggregates. Exposure to heavy metals such as Cd, Hg, Mn, and Pb is known to increase Aß production, and these metals bind to Aß peptides and modulate their aggregation. The possible effects of U in AD pathology have been sparsely studied. Here, we use biophysical techniques to study in vitro interactions between Aß peptides and uranyl ions, UO22+, of DU. We show for the first time that uranyl ions bind to Aß peptides with affinities in the micromolar range, induce structural changes in Aß monomers and oligomers, and inhibit Aß fibrillization. This suggests a possible link between AD and U exposure, which could be further explored by cell, animal, and epidemiological studies. General toxic mechanisms of uranyl ions could be modulation of protein folding, misfolding, and aggregation.

Redox and metal ion binding properties of human insulin-like growth factor 1 determined by electrospray ionization mass spectrometry.

Insulin-like growth factor 1 (IGF-1) is a 70-residue hormone containing three intramolecular disulfide bridges. IGF-1 and other growth factors are oxidatively folded in the endoplasmic reticulum and act primarily in the blood, under relatively oxidative conditions. It is known that IGF-1 exists in various intracellular and extracellular compartments in the oxidized form; however, the reduction potential of IGF-1 and the ability of fully reduced IGF-1, which contains six cysteine residues, to bind transition metal ions are not known. In this work, we determine that the redox potential of human IGF-1 is equal to -332 mV and the reduced form of hIGF-1 can bind cooperatively four Cu(+) ions, most probably into a tetracopper-hexathiolate cluster. The Cu(+) binding affinity of hIGF-1 is, however, approximately 3 times lower than that for the copper chaperones; thus, we can conclude that fully reduced hIGF-1 cannot compete with known Cu(+)-binding proteins.

Interference of low-molecular substances with the thioflavin-T fluorescence assay of amyloid fibrils.

Abnormal fibrillization of amyloidogenic peptides/proteins has been linked to various neurodegenerative diseases such as Alzheimer's and Parkinson's disease as well as with type-II diabetes mellitus. The kinetics of protein fibrillization is commonly studied by using a fluorescent dye Thioflavin T (ThT) that binds to protein fibrils and exerts increased fluorescence intensity in bound state. Recently, it has been demonstrated that several low-molecular weight compounds like Basic Blue 41, Basic Blue 12, Azure C, and Tannic acid interfere with the fluorescence of ThT bound to Alzheimers' amyloid-ß fibrils and cause false positive results during the screening of fibrillization inhibitors. In the current study, we demonstrated that the same selected substances also decrease the fluorescence signal of ThT bound to insulin fibrils already at submicromolar or micromolar concentrations. Kinetic experiments show that unlike to true inhibitors, these compounds did neither decrease the fibrillization rate nor increase the lag-period. Absence of soluble insulin in the end of the experiment confirmed that these compounds do not disaggregate the insulin fibrils and, thus, are not fibrillization inhibitors at concentrations studied. Our results show that interference with ThT test is a general phenomenon and more attention has to be paid to interpretation of kinetic results of protein fibrillization obtained by using fluorescent dyes.

α-Lipoic Acid Has the Potential to Normalize Copper Metabolism, Which Is Dysregulated in Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is an age-dependent progressive neurodegenerative disorder and the most common cause of dementia. The treatment and prevention of AD present immense yet unmet needs. One of the hallmarks of AD is the formation of extracellular amyloid plaques in the brain, composed of amyloid-ß (Aß) peptides. Besides major amyloid-targeting approach there is the necessity to focus also on alternative therapeutic strategies. One factor contributing to the development of AD is dysregulated copper metabolism, reflected in the intracellular copper deficit and excess of extracellular copper. OBJECTIVE: In the current study, we follow the widely accepted hypothesis that the normalization of copper metabolism leads to the prevention or slowing of the disease and search for new copper-regulating ligands. METHODS: We used cell culture, ICP MS, and Drosophila melanogaster models of AD. RESULTS: We demonstrate that the natural intracellular copper chelator, α-lipoic acid (LA) translocates copper from extracellular to intracellular space in an SH-SY5Y-based neuronal cell model and is thus suitable to alleviate the intracellular copper deficit characteristic of AD neurons. Furthermore, we show that supplementation with LA protects the Drosophila melanogaster models of AD from developing AD phenotype by improving locomotor activity of fruit fly with overexpression of human Aß with Iowa mutation in the fly brain. In addition, LA slightly weakens copper-induced smooth eye phenotype when amyloid-ß protein precursor (AßPP) and beta-site AßPP cleaving enzyme 1 (BACE1) are overexpressed in eye photoreceptor cells. CONCLUSION: Collectively, these results provide evidence that LA has the potential to normalize copper metabolism in AD.

Mercury Ion Binding to Apolipoprotein E Variants ApoE2, ApoE3, and ApoE4: Similar Binding Affinities but Different Structure Induction Effects.

Mercury intoxication typically produces more severe outcomes in people with the APOE-ε4 gene, which codes for the ApoE4 variant of apolipoprotein E, compared to individuals with the APOE-ε2 and APOE-ε3 genes. Why the APOE-ε4 allele is a risk factor in mercury exposure remains unknown. One proposed possibility is that the ApoE protein could be involved in clearing of heavy metals, where the ApoE4 protein might perform this task worse than the ApoE2 and ApoE3 variants. Here, we used fluorescence and circular dichroism spectroscopies to characterize the in vitro interactions of the three different ApoE variants with Hg(I) and Hg(II) ions. Hg(I) ions displayed weak binding to all ApoE variants and induced virtually no structural changes. Thus, Hg(I) ions appear to have no biologically relevant interactions with the ApoE protein. Hg(II) ions displayed stronger and very similar binding affinities for all three ApoE isoforms, with K D values of 4.6 µM for ApoE2, 4.9 µM for ApoE3, and 4.3 µM for ApoE4. Binding of Hg(II) ions also induced changes in ApoE superhelicity, that is, altered coil-coil interactions, which might modify the protein function. As these structural changes were most pronounced in the ApoE4 protein, they could be related to the APOE-ε4 gene being a risk factor in mercury toxicity.

Interaction between oligomers of stefin B and amyloid-beta in vitro and in cells.

To contribute to the question of the putative role of cystatins in Alzheimer disease and in neuroprotection in general, we studied the interaction between human stefin B (cystatin B) and amyloid-beta-(1-40) peptide (Abeta). Using surface plasmon resonance and electrospray mass spectrometry we were able to show a direct interaction between the two proteins. As an interesting new fact, we show that stefin B binding to Abeta is oligomer specific. The dimers and tetramers of stefin B, which bind Abeta, are domain-swapped as judged from structural studies. Consistent with the binding results, the same oligomers of stefin B inhibit Abeta fibril formation. When expressed in cultured cells, stefin B co-localizes with Abeta intracellular inclusions. It also co-immunoprecipitates with the APP fragment containing the Abeta epitope. Thus, stefin B is another APP/Abeta-binding protein in vitro and likely in cells.

Zn(II) ions co-secreted with insulin suppress inherent amyloidogenic properties of monomeric insulin.

Insulin, a 51-residue peptide hormone, is an intrinsically amyloidogenic peptide, forming amyloid fibrils in vitro. In the secretory granules, insulin is densely packed together with Zn(II) into crystals of Zn(2)Insulin(6) hexamer, which assures osmotic stability of vesicles and prevents fibrillation of the peptide. However, after release from the pancreatic beta-cells, insulin dissociates into active monomers, which tend to fibrillize not only at acidic, but also at physiological, pH values. The effect of co-secreted Zn(II) ions on the fibrillation of monomeric insulin is unknown, however, it might prevent insulin fibrillation. We showed that Zn(II) inhibits fibrillation of monomeric insulin at physiological pH values by forming a soluble Zn(II)-insulin complex. The inhibitory effect of Zn(II) ions is very strong at pH 7.3 (IC(50)=3.5 microM), whereas at pH 5.5 it progressively weakens, pointing towards participation of the histidine residue(s) in complex formation. The results obtained indicate that Zn(II) ions might suppress fibrillation of insulin at its release sites and in circulation. It is hypothesized that misfolded oligomeric intermediates occurring in the insulin fibrillation pathway, especially in zinc-deficient conditions, might induce autoantibodies against insulin, which leads to beta-cell damage and autoimmune Type 1 diabetes.

Mitochondrial copper(I) transfer from Cox17 to Sco1 is coupled to electron transfer.

The human protein Cox17 contains three pairs of cysteines. In the mitochondrial intermembrane space (IMS) it exists in a partially oxidized form with two S-S bonds and two reduced cysteines (HCox17(2S-S)). HCox17(2S-S) is involved in copper transfer to the human cochaperones Sco1 and Cox11, which are implicated in the assembly of cytochrome c oxidase. We show here that Cu(I)HCox17(2S-S), i.e., the copper-loaded form of the protein, can transfer simultaneously copper(I) and two electrons to the human cochaperone Sco1 (HSco1) in the oxidized state, i.e., with its metal-binding cysteines forming a disulfide bond. The result is Cu(I)HSco1 and the fully oxidized apoHCox17(3S-S), which can be then reduced by glutathione to apoHCox17(2S-S). The HSco1/HCox17(2S-S) redox reaction is thermodynamically driven by copper transfer. These reactions may occur in vivo because HSco1 can be found in the partially oxidized state within the IMS, consistent with the variable redox properties of the latter compartment. The electron transfer-coupled metallation of HSco1 can be a mechanism within the IMS for an efficient specific transfer of the metal to proteins, where metal-binding thiols are oxidized. The same reaction of copper-electron-coupled transfer does not occur with the human homolog of Sco1, HSco2, for kinetic reasons that may be ascribed to the lack of a specific metal-bridged protein-protein complex, which is instead observed in the Cu(I)HCox17(2S-S)/HSco1 interaction.

Label-free high-throughput screening assay for inhibitors of Alzheimer's amyloid-ß peptide aggregation based on MALDI MS.

Aggregation of amyloid-ß (Aß) peptides is causatively linked to Alzheimer's disease (AD); thus, suppression of this process by small molecule inhibitors is a widely accepted therapeutic and preventive strategy for AD. Screening of the inhibitors of Aß aggregation deserves much attention; however, despite intensive efforts, there are only a few high-throughput screening methods available, all of them having drawbacks related to the application of external fluorescent probes or artificial Aß derivatives. We have developed a label-free MALDI MS-based screening test for inhibitors of Aß42 fibrillization that exhibits high sensitivity, speed, and automation possibilities suitable for high-throughput screening. The test was evaluated by transmission electron microscopy and compared with a fluorimetric thioflavin-based assay, where interference of a number of tested compounds with thioflavin T binding and/or fluorescence caused false-positive results. The MALDI MS-based method can significantly speed up in vitro screening of compound libraries for inhibitors of Aß42 fibrillization.

Copper(II) partially protects three histidine residues and the N-terminus of amyloid-ß peptide from diethyl pyrocarbonate (DEPC) modification.

Diethyl pyrocarbonate (DEPC) has been primarily used as a residue-specific modifying agent to study the role of His residues in peptide/protein and enzyme function; however, its action is not specific, and several other residues can also be modified. In the current study, we monitored the reaction of DEPC with amyloid-beta (Aß) peptides and insulin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and determined the modification sites by electrospray ionization quadrupole time-of-flight tandem MS (ESI Q-TOF MS/MS). Our results indicate that five residues in Aß1-42 are modified in the presence of 30-fold molar excess of DEPC. After hydroxylamine treatment (which removes modifications from three His residues), two labels remain bound at the peptide N terminus and Lys16. DEPC treatment of Aß1-42 promotes peptide aggregation, as monitored through the loss of soluble Aß42 in a semi-quantitative MALDI-TOF MS assay. It has been previously proposed that Cu(II) ions protect Aß1-16 from DEPC modification through binding to His6. We confirmed that Cu(II) ions decrease the stoichiometry of Aß1-16 modification with the excess of DEPC being lower as compared to the control, which indicates that Cu(II) protects Aß from DEPC modification. Sequencing of obtained Cu(II)-protected Aß1-16 samples showed that Cu(II) does not protect any residues completely, but partially protects all three His residues and the N terminus. Thus, the protection by Cu(II) ions is not related to specific metal binding to a particular residue (e.g. His6), but rather all His residues and the N terminus are involved in binding of Cu(II) ions. These results allow us to elucidate the effect of DEPC modification on amyloidogenity of human Aß and to speculate about the role of His residues in these processes.

Copper(II)-binding equilibria in human blood.

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%).