The Angiotensin Metabolite His-Leu Is a Strong Copper Chelator Forming Highly Redox Active Species.

His-Leu is a hydrolytic byproduct of angiotensin metabolism, whose concentration in the bloodstream could be at least micromolar. This encouraged us to investigate its Cu(II) binding properties and the concomitant redox reactivity. The Cu(II) binding constants were derived from isothermal titration calorimetry and potentiometry, while identities and structures of complexes were obtained from ultraviolet-visible, circular dichroism, and room-temperature electronic paramagnetic resonance spectroscopies. Four types of Cu(II)/His-Leu complexes were detected. The histamine-like complexes prevail at low pH. At neutral and mildly alkaline pH and low Cu(II):His-Leu ratios, they are superseded by diglycine-like complexes involving the deprotonated peptide nitrogen. At His-Leu:Cu(II) ratios of ≥2, bis-complexes are formed instead. Above pH 10.5, a diglycine-like complex containing the equatorially coordinated hydroxyl group predominates at all ratios tested. Cu(II)/His-Leu complexes are also strongly redox active, as demonstrated by voltammetric studies and the ascorbate oxidation assay. Finally, numeric competition simulations with human serum albumin, glycyl-histydyl-lysine, and histidine revealed that His-Leu might be a part of the low-molecular weight Cu(II) pool in blood if its abundance is >10 µM. These results yield further questions, such as the biological relevance of ternary complexes containing His-Leu.

Concentrations of Transition Metal Ions in Rat Lungs after Tobacco Smoke Exposure and Treatment with His-Leu Dipeptide.

Tobacco smoking is deleterious to the lungs because it exposes them to many toxic substances. These include transition metal ions, such as cadmium. However, there is a lack of information about the influence of endogenous metal-binding peptides, such as His-Leu (HL), on the lung distribution of transition metals in smokers. To address this, we administered HL subcutaneously to rats exposed to tobacco smoke for six weeks, then we measured the concentrations of transition metal ions in the lungs. We found that exposure to tobacco smoke elevates the concentrations of Cd(II) and Cu(II). Administration of the HL peptide, whose elevation is a consequence of angiotensin receptor blocker anti-hypertension therapy, increases the concentration of Fe in the lungs of rats exposed to smoke. These findings suggest that smoking is a risk factor for patients receiving angiotensin receptor blockers to treat hypertension.

Ni(II) Ions May Target the Entire Melatonin Biosynthesis Pathway-A Plausible Mechanism of Nickel Toxicity.

Nickel is toxic to humans. Its compounds are carcinogenic. Furthermore, nickel allergy is a severe health problem that affects approximately 10-20% of humans. The mechanism by which these conditions develop remains unclear, but it may involve the cleavage of specific proteins by nickel ions. Ni(II) ions cleave the peptide bond preceding the Ser/Thr-Xaa-His sequence. Such sequences are present in all four enzymes of the melatonin biosynthesis pathway, i.e., tryptophan 5-hydroxylase 1, aromatic-l-amino-acid decarboxylase, serotonin N-acetyltransferase, and acetylserotonin O-methyltransferase. Moreover, fragments prone to Ni(II) are exposed on surfaces of these proteins. Our results indicate that all four studied fragments undergo cleavage within tens of hours at pH 8.2 and 37 °C, corresponding with the conditions in the mitochondrial matrix. Since melatonin, a potent antioxidant and anti-inflammatory agent, is synthesized within the mitochondria of virtually all human cells, depleting its supply may be detrimental, e.g., by raising the oxidative stress level. Intriguingly, Ni(II) ions have been shown to mimic hypoxia through the stabilization of HIF-1α protein, but melatonin prevents the action of HIF-1α. Considering all this, the enzymes of the melatonin biosynthesis pathway seem to be a toxicological target for Ni(II) ions.

Phosphorylation Impacts Cu(II) Binding by ATCUN Motifs.

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.

Ternary Cu2+ Complexes of Human Serum Albumin and Glycyl-l-histidyl-l-lysine.

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.

Intermediate Cu(II)-Thiolate Species in the Reduction of Cu(II)GHK by Glutathione: A Handy Chelate for Biological Cu(II) Reduction.

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.

Cu(II)-Binding N-Terminal Sequences of Human Proteins.

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.

Design and in Vitro Characterization of Tricyclic Benzodiazepine Derivatives as Potent and Selective Antileukemic Agents.

Currently available chemotherapeutic treatments for blood cancers (leukemia) usually have strong side effects. More selective, efficient, and less toxic anticancer agents are needed. We synthesized seven, new, optically pure (12aS)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione derivatives and examined their cytotoxicity towards eight cancer cell lines, including urinary bladder (TCC-SUP, UM-UC-3, KU-19-9), colon (LoVo), and breast (MCF-7, MDA-MB-231) cancer representatives, as well as two leukemic cell lines (MV-4-11, CCRF-CEM) and normal murine fibroblasts (Balb/3T3) as reference cell line. Three of the seven newly-obtained compounds ((12aS)-8-bromo-2-(3-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione, (12aS)-8,9-dimethoxy-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione and (12aS)-8-nitro-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione, showed enhanced activity and selectivity toward the leukemic MV-4-11 cell lines when compared to our previously reported compounds, with IC50 values in the range of 2.9-5.6 µM. Additionally, (12aS)-9-nitro-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione exhibited a strong cytotoxic effect against the leukemic CCRF-CEM (IC50 =6.1 µM) and MV-4-11 (IC50 =11.0 µM) cell lines, a moderate cytotoxic effect toward other tumor lines (IC50 =31.8-55.0 µM) and very weak cytotoxic effect toward the Balb/3T3 reference cell lines. Selected compounds were further evaluated for their potential to induce apoptotic cell death in MV-4-11 cells by measuring caspase-3 activity. We also established the crystal structure of three products and investigated the effect of 22 derivatives of 1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione on the activity of the cancer-associated enzyme autotaxin. All compounds proved to be weak inhibitors of autotaxin, although some (R) and (S) enantiomers had Ki values of 10-19 µM. The obtained results showed that the tested compounds exhibited a selective antileukemic effect, which appeared not to be related directly to autotaxin. Molecular targets responsible for this effect remain to be identified. The newly obtained compounds can be used in the search for new, selective anticancer therapies.

The Sub-picomolar Cu2+ Dissociation Constant of Human Serum Albumin.

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 cKCuCu(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.

Peptide Bond Cleavage by Ni(II) Ions within the Nuclear Localization Signal Sequence.

Nickel is harmful to humans, being both carcinogenic and allergenic. However, the mechanisms of this toxicity are still unresolved. We propose that Ni(II) ions disintegrate proteins by hydrolysis of peptide bonds preceding the Ser/Thr-Xaa-His sequences. Such sequences occur in nuclear localization signals (NLSs) of human phospholipid scramblase 1, Sam68-like mammalian protein 2, and CLK3 kinase. We performed spectroscopic experiments showing that model nonapeptides derived from these NLSs bind Ni(II) at physiological pH. We also proved that these sequences are prone to Ni(II) hydrolysis. Thus, the aforementioned NLSs may be targets for nickel toxicity. This implies that Ni(II) ions disrupt the transport of some proteins from cytoplasm to cell nucleus.

Ternary Cu(II) Complex with GHK Peptide and Cis-Urocanic Acid as a Potential Physiologically Functional Copper Chelate.

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 Palladium(II) Complex of Aß4-16 as Suitable Model for Structural Studies of Biorelevant Copper(II) Complexes of N-Truncated Beta-Amyloids.

The Aß4-42 peptide is a major beta-amyloid species in the human brain, forming toxic aggregates related to Alzheimer's Disease. It also strongly chelates Cu(II) at the N-terminal Phe-Arg-His ATCUN motif, as demonstrated in Aß4-16 and Aß4-9 model peptides. The resulting complex resists ROS generation and exchange processes and may help protect synapses from copper-related oxidative damage. Structural characterization of Cu(II)Aß4-x complexes by NMR would help elucidate their biological function, but is precluded by Cu(II) paramagneticism. Instead we used an isostructural diamagnetic Pd(II)-Aß4-16 complex as a model. To avoid a kinetic trapping of Pd(II) in an inappropriate transient structure, we designed an appropriate pH-dependent synthetic procedure for ATCUN Pd(II)Aß4-16, controlled by CD, fluorescence and ESI-MS. Its assignments and structure at pH 6.5 were obtained by TOCSY, NOESY, ROESY, 1H-13C HSQC and 1H-15N HSQC NMR experiments, for natural abundance 13C and 15N isotopes, aided by corresponding experiments for Pd(II)-Phe-Arg-His. The square-planar Pd(II)-ATCUN coordination was confirmed, with the rest of the peptide mostly unstructured. The diffusion rates of Aß4-16, Pd(II)-Aß4-16 and their mixture determined using PGSE-NMR experiment suggested that the Pd(II) complex forms a supramolecular assembly with the apopeptide. These results confirm that Pd(II) substitution enables NMR studies of structural aspects of Cu(II)-Aß complexes.

Thymidylate synthase-catalyzed, tetrahydrofolate-dependent self-inactivation by 5-FdUMP.

In view of previous crystallographic studies, N4-hydroxy-dCMP, a slow-binding thymidylate synthase inhibitor apparently caused "uncoupling" of the two thymidylate synthase-catalyzed reactions, including the N5,10-methylenetetrahydrofolate one-carbon group transfer and reduction, suggesting the enzyme's capacity to use tetrahydrofolate as a cofactor reducing the pyrimidine ring C(5) in the absence of the 5-methylene group. Testing the latter interpretation, a possibility was examined of a TS-catalyzed covalent self-modification/self-inactivation with certain pyrimidine deoxynucleotides, including 5-fluoro-dUMP and N4-hydroxy-dCMP, that would be promoted by tetrahydrofolate and accompanied with its parallel oxidation to dihydrofolate. Electrophoretic analysis showed mouse recombinant TS protein to form, in the presence of tetrahydrofolate, a covalently bound, electrophoretically separable 5-fluoro-dUMP-thymidylate synthase complex, similar to that produced in the presence of N5,10-methylenetetrahydrofolate. Further studies of the mouse enzyme binding with 5-fluoro-dUMP/N4-hydroxy-dCMP by TCA precipitation of the complex on filter paper showed it to be tetrahydrofolate-promoted, as well as to depend on both time in the range of minutes and the enzyme molecular activity, indicating thymidylate synthase-catalyzed reaction to be responsible for it. Furthermore, the tetrahydrofolate- and time-dependent, covalent binding by thymidylate synthase of each 5-fluoro-dUMP and N4-hydroxy-dCMP was shown to be accompanied by the enzyme inactivation, as well as spectrophotometrically confirmed dihydrofolate production, the latter demonstrated to depend on the reaction time, thymidylate synthase activity and temperature of the incubation mixture, further documenting its catalytic character.

Interplay between Copper, Neprilysin, and N-Truncation of ß-Amyloid.

Sporadic Alzheimer's disease (AD) is associated with an inefficient clearance of the ß-amyloid (Aß) peptide from the central nervous system. The protein levels and activity of the Zn2+-dependent endopeptidase neprilysin (NEP) inversely correlate with brain Aß levels during aging and in AD. The present study considered the ability of Cu2+ ions to inhibit human recombinant NEP and the role for NEP in generating N-truncated Aß fragments with high-affinity Cu2+ binding motifs that can prevent this inhibition. Divalent copper noncompetitively inhibited NEP ( Ki = 1.0 µM),  while proteolysis of Aß yielded the soluble, Aß4-9 fragment that can bind Cu2+ with femtomolar affinity at pH 7.4. This provides Aß4-9 with the potential to act as a Cu2+ carrier and to mediate its own production by preventing NEP inhibition. Enzyme inhibition at high Zn2+ concentrations ( Ki = 20 µM) further suggests a mechanism for modulating NEP activity, Aß4-9 production, and Cu2+ homeostasis.

Gly-His-Thr-Asp-Amide, an Insulin-Activating Peptide from the Human Pancreas Is a Strong Cu(II) but a Weak Zn(II) Chelator.

The Cu(II) and Zn(II) binding abilities of Gly-His-Thr-Asp-amide (GHTD-am), a tetrapeptide coreleased from the pancreas along with insulin, were studied using UV-vis and circular dichroism spectroscopies, potentiometry, and calorimetry. GHTD-am is a very strong Cu(II) chelator, forming a three-nitrogen complex with a conditional affinity constant C K at pH 7.4 of 4.5 × 1012 M-1. The fourth coordination site can be occupied by a solvent molecule or a ternary ligand, such as imidazole, with C K on the order of several hundred reciprocal molar. The Zn(II) binding ability of GHTD-am is relatively weak, with C K values at pH 7.4 of 3.0 × 104 and 2.0 × 103 M-1 for the first and second GHTD-am molecule coordinated, respectively. These results are discussed in light of the modes of interactions of Zn(II) and Cu(II) ions with insulin. A direct effect of GHTD-am on the Zn(II) interactions with insulin is unlikely, but its Cu(II) complex may have a biological relevance because of its high affinity and ability to form ternary complexes.

Properties of phosphorylated thymidylate synthase.

Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent.

Unusual Zn(II) Affinities of Zinc Fingers of Poly(ADP-ribose)Polymerase 1 (PARP-1) Nuclear Protein.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a key eukaryotic enzyme,catalyzing the NAD+ dependent poly(ADP-ribosyl)ation of protein substrates, crucial for major DNA repair pathways, and involved in other fundamental cellular processes, such as transcription, cell cycle control, and apoptosis. Its ability to bind DNA depends on two CCHC zinc finger domains, in short, PARPzf1 and PARPzf2. Using spectroscopic methods and competitive titrations with Zn(II), Co(II), and Ni(II) ions, we determined conditional dissociation constants for Zn(II) complexes of PARPzf1 and PARPzf2 at pH 7.4 (HEPESbuffer) as 26 ± 4 nM and 4 ± 1 pM, respectively. The former value indicates an extremely low affinity of PARPzf1 toward metal ions, meaning that under cellular conditions PARP1zf might be largely present in a "metal-free" state. This finding provides a clue to the high susceptibility of PARP-1 to oxidative stress but also raises questions regarding the activation of PARPzf1 under cellular conditions. We also determined conditional dissociation constants for Ni(II) complexes of PARPzf1 and PARPzf2 under the same conditions as 0.78 ± 0.04 µM and 0.26 ± 0.05 nM, respectively.

A Functional Role for Aß in Metal Homeostasis? N-Truncation and High-Affinity Copper Binding.

Accumulation of the ß-amyloid (Aß) peptide in extracellular senile plaques rich in copper and zinc is a defining pathological feature of Alzheimer's disease (AD). The Aß1-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 Aß4-42 peptide is a major Aß 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 Aß1-x peptides. By using Aß4-16 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 Aß4-16 and Aß4-42 possess negligible redox activity. Combined with the predominance of Aß4-42 in the brain, our results suggest a physiological role for this isoform in metal homeostasis within the central nervous system.

Human annexins A1, A2, and A8 as potential molecular targets for Ni(II) ions.

Nickel is harmful for humans, but molecular mechanisms of its toxicity are far from being fully elucidated. One of such mechanisms may be associated with the Ni(II)-dependent peptide bond hydrolysis, which occurs before Ser/Thr in Ser/Thr-Xaa-His sequences. Human annexins A1, A2, and A8, proteins modulating the immune system, contain several such sequences. To test if these proteins are potential molecular targets for nickel toxicity we characterized the binding of Ni(II) ions and hydrolysis of peptides Ac-KALTGHLEE-am (A1-1), Ac-TKYSKHDMN-am (A1-2), and Ac-GVGTRHKAL-am (A1-3), from annexin A1, Ac-KMSTVHEIL-am (A2-1) and Ac-SALSGHLET-am (A2-2), from annexin A2, and Ac-VKSSSHFNP-am (A8-1), from annexin A8, using UV-vis and circular dichroism (CD) spectroscopies, potentiometry, isothermal titration calorimetry, high-performance liquid chromatography (HPLC), and electrospray ionization mass spectrometry (ESI-MS). We found that at physiological conditions (pH 7.4 and 37 °C) peptides A1-2, A1-3, A8-1, and to some extent A2-2 bind Ni(II) ions sufficiently strongly in 4N complexes and are hydrolyzed at sufficiently high rates to justify the notion that these annexins can undergo nickel hydrolysis in vivo. These results are discussed in the context of specific biochemical interactions of respective proteins. Our results also expand the knowledge about Ni(II) binding to histidine peptides by determination of thermodynamic parameters of this process and spectroscopic characterization of 3N complexes. Altogether, our results indicate that human annexins A1, A2, and A8 are potential molecular targets for nickel toxicity and help design appropriate cellular studies.

Revised coordination model and stability constants of Cu(II) complexes of tris buffer.

2-Amino-2-hydroxymethyl-propane-1,3-diol, or tris(hydroxymethyl)aminomethane (Tris), is probably the most common biochemical buffer used alone or in combination with other buffers because it is stable, unreactive, and compatible with most proteins and other biomolecules. Being nontoxic, it has even found applications in medicine. Tris is known, however, to coordinate transition metal ions, Cu(II) among them. Although often ignored, this feature affects interactions of Cu(II) ions with biomolecules, as Tris is usually used in high molar excess. Therefore, it is important to have precise knowledge on the stoichiometry, stability, and reactivity of cupric Tris complexes. The literature data are incoherent in this respect. We reinvestigated the complex formation in the Tris-Cu(II) system by potentiometry, UV-vis, ESI-MS, and EPR at a broad range of concentrations and ratios. We found, contrary to several previous papers, that the maximum stoichiometry of Tris to Cu(II) is 2 and at neutral pH, dimeric complexes are formed. The apparent affinity of Tris buffer for Cu(II), determined by the competitivity index (CI) approach [Krezel, A.; Wójcik, J.; Maciejczyk, M.; Bal, W. Chem. Commun. 2003, 6, 704-705] at pH 7.4 varies between 2 × 10(6) and 4 × 10(4) M(-1), depending on the Tris and Cu(II) concentrations and molar ratio.