Milk ceruloplasmin is a valuable source of nutrient copper ions for mammalian newborns

This research focuses on the role of milk ceruloplasmin (Cp), the main extracellular copper-containing protein of vertebrates, as a source of copper for newborns. In the first part of the study, Cp concentration and Cp-associated copper were measured in human skimmed milk at the 1st and the 5th days postpartum. It was shown that most of the copper was associated with Cp and that the decrease in copper concentration during lactation was related to the drop of Cp levels. The following in vivo experiments demonstrated that milk [(125)I]Cp per os administered to 6-day-old rats (embryonic-type copper metabolism) was transported into their bloodstream. The electrophoretic mobility and relative molecular weight of [(125)I]Cp transferred through the cellular barrier remained unaltered. However, 22-day-old rats (adult-type copper metabolism) digested the administered milk [(125)I]Cp completely. In the final part of the study, newborn rats were fed with baby formula for 8d. It was found that these rats switched their copper metabolism from embryonic type to adult type earlier than their littermates fed by dams. Activation of Cp gene expression in the liver, increased Cp and copper concentrations in the blood, and reduced copper content of the liver were observed in the rats fed with baby formula. In the brain, no copper concentration change was observed, but Cp and copper concentrations were dramatically increased in the cerebrospinal fluid. The role of milk Cp as a source of copper adapted to embryonic-type copper metabolism is discussed.     

On the lability and functional significance of the type 1 copper pool in ceruloplasmin

 The possibility that ceruloplasmin (CP) functions as a copper transferase has fueled a continuing interest in studies of the copper release process. The principal goal of the current investigation has been to identify the most labile copper centers in sheep protein. In fact, subjecting the enzyme to a slow flux of cyanide at pH 5.2 under nitrogen in the presence of ascorbate and a phenanthroline ligand produces partially demetalated forms of the protein. By standard chromatographic techniques it is possible to isolate protein with a Cu/CP ratio of ∼4 or ∼5 as opposed to the native protein which has Cu/CP=5.8. In contrast to other blue oxidases, analysis suggests that CP preferentially loses its type 1 coppers under these conditions. Thus, the spectroscopic signals from the type 1 centers exhibit a loss of intensity while the EPR signal of the type 2 copper becomes stronger. Furthermore, the Cu/CP≈4 and Cu/CP≈5 components retain about 50% of the activity of the native protein, consistent with an intact type 2/type 3 cluster. All three type 1 copper sites appear to suffer copper loss. Reconstitution with a copper(I) reagent restores the spectroscopic properties of the native protein and 90% of the original activity. The results suggest a possible functional significance for the presence of three type 1 coppers in CP. By employing a pool of redox-active but relatively labile type 1 copper centers, the enzyme can serve as a copper donor, if necessary, without completely sacrificing its oxidase activity. Received: 15 February 1999 / Accepted: 22 April 1999     

Intracellular transport and degradation of I-labelled denatured serum albumin in isolated nonparenchymal rat liver cells

The intracellular movement, following uptake of ¹²⁵I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker β-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker β-acetylglucosaminidase, suggesting lysosomal degradation.     

A comparative study of plasma membrane Mg2+-ATPase activities in normal,regenerating and malignant cells

Plasma membranes have been prepared from rat normal liver cells, regenerating liver cells and Yoshida ascites hepatoma 66 cells after intact cells were first bound to polylysine-coated polyacrylamide beads, and the membrane-associated Mg²⁺-ATPase activity was assayed directly on beads with membrane attached. With plasma membranes from normal liver cells, K_m for ATP and V were found to be higher than those in regenerating liver cells and hepatoma cells. Vanadate caused a different sensitivity of the activity, without an effect in normal liver cells and with an inhibition in regenerating liver cells and hepatoma cells. The activity in normal and regenerating liver cells decreased with increasing temperature above 24–30°C, while the activity in hepatoma cells continued to increase linearly to 37°C. Unlike the enzyme in normal and regenerating liver cells, the hepatoma enzyme was shown to have a higher phase transition temperature and lower activation energies. In all three kinds of cells the activity was increased by the dephosphorylation of plasma membranes and unaffected by the phosphorylation. By means of histochemical Mg²⁺-ATPase staining applied on polyacrylamide gels, at least three major bands which show the enzymic activity were visible in normal and regenerating liver and a single band was detected in hepatoma cells.     

Expression of metallothionein in the liver and kidney of rats is influenced by excess dietary histidine

It is well known that excess dietary histidine induces the metabolic changes in copper and zinc. Therefore, this study was carried out to clarify whether excess dietary histidine alters the gene expressions of metallothionein-1 and metallothionein-2 in the liver and kidney. Male rats were fed the control (ad libitum and pair-fed) or histidine-excess (50 g of L-histidine per kg of diet) diet for 0, 1 and 3 days. The levels of liver metallothionein-1 and metallothionein-2 mRNA were markedly lower in the rats fed the histidine-excess diet as compared to those of the control (ad libitum and pair-fed) diet, when fed for 1 or 3 days. The levels of renal metallothionein-1 and metallothionein-2 mRNA in the rats fed the histidine-excess diet were higher or tended to be higher as compared with the rats fed the control (ad libitum and pair-fed) diet when fed for 1 or 3 days, respectively. At the same time, hepatic copper content was decreased and renal zinc content was increased by dietary histidine. It thus appears, that such a response on the level of liver metallothionein mRNA might be related to the contents of liver copper, but of kidney metallothionein mRNA might be due to the content of zinc.     

Homoleptic copper(II) and copper(I) complexes of 5,6-dihydro-5,6-epoxy-1,10-phenanthroline. Six-coordinate copper(I) in solution

Reaction of 5,6-dihydro-5,6-epoxy-1,10-phenanthroline (L) with Cu(ClO₄)₂·6H₂O in methanol in 3:1 M ratio at room temperature yields light green [CuL₃](ClO₄)₂·H₂O (1). The X-ray crystal structure of the hemi acetonitrile solvate [CuL₃](ClO₄)₂·0.5CH₃CN has been determined which shows Jahn-Teller distortion in the CuN₆ core present in the cation [CuL₃]²⁺. Complex 1 gives an axial EPR spectrum in acetonitrile-toluene glass with g_|| = 2.262 (A_|| = 169 × 10⁻⁴ cm⁻¹) and g_⊥ = 2.069. The Cu(II/I) potential in 1 in CH₂Cl₂ at a glassy carbon electrode is 0.32 V versus NHE. This potential does not change with the addition of extra L in the medium implicating generation of a six-coordinate copper(I) species [CuL₃]⁺ in solution. B3LYP/LanL2DZ calculations show that the six Cu-N bond distances in [CuL₃]⁺ are 2.33, 2.25, 2.32, 2.25, 2.28 and 2.25 Å while the ideal Cu(I)-N bond length in a symmetric Cu(I)N₆ moiety is estimated as 2.25 Å. Reaction of L with Cu(CH₃CN)₄ClO₄ in dehydrated methanol at room temperature even in 4:1 M proportion yields [CuL₂]ClO₄ (2). Its ¹H NMR spectrum indicates that the metal in [CuL₂]⁺ is tetrahedral. The Cu(II/I) potential in 2 is found to be 0.68 V versus NHE in CH₂Cl₂ at a glassy carbon electrode. In presence of excess L, 2 yields the cyclic voltammogram of 1. From ¹H NMR titration, the free energy of binding of L to [CuL₂]⁺ to produce [CuL₃]⁺ in CD₂Cl₂ at 298 K is estimated as −11.7 (±0.2) kJ mol⁻¹.     

Application of saccharose as copper(II) ligand for electroless copper plating solutions

Saccharose, forming sufficiently stable complexes with copper(II) ions in alkaline solutions, was found to be a suitable ligand for copper(II) chelating in alkaline (pH>12) electroless copper deposition solutions. Reduction of copper(II)-saccharose complexes by hydrated formaldehyde was investigated and the copper deposits formed were characterized. The thickness of the compact copper coatings obtained under optimal operating conditions in 1h reaches ca. 2 microm at ambient temperature. The plating solutions were stable and no signs of Cu(II) reduction in the bulk solution were observed. Results were compared with those systems operating with other copper(II) ligands.     

Optimization of an enrichment process for circulating tumor cells from the blood of head and neck cancer patients through depletion of normal cells

The optimization of a purely negative depletion, enrichment process for circulating tumor cells (CTCs) in the peripheral blood of head and neck cancer patients is presented. The enrichment process uses a red cell lysis step followed by immunomagnetic labeling, and subsequent depletion, of CD45 positive cells. A number of relevant variables are quantified, or attempted to be quantified, which control the performance of the enrichment process. Six different immunomagnetic labeling combinations were evaluated as well as the significant difference in performance with respect to the blood source: buffy coats purchased from the Red Cross, fresh, peripheral blood from normal donors, and fresh peripheral blood from human cancer patients. After optimization, the process is able to reduce the number of normal blood cells in a cancer patient's blood from 4.05 × 10⁹ to 8.04 × 10³ cells/mL and still recover, on average, 2.32 CTC per mL of blood. For all of the cancer patient blood samples tested in which CTC were detected (20 out of 26 patients) the average recovery of CTCs was 21.7 per mL of blood, with a range of 282 to 0.53 CTC. Since the initial number of CTC in a patient's blood is unknown, and most probably varies from patient to patient, the recovery of the CTC is unknown. However, spiking studies of a cancer cell line into normal blood, and subsequent enrichment using the optimized protocol indicated an average recovery of approximately 83%. Unlike a majority of other published studies, this study focused on quantifying as many factors as possible to facilitate both the optimization of the process as well as provide information for current and future performance comparisons. The authors are not aware any other reported study which has achieved the performance reported here (a 5.66 log₁₀) in a purely negative enrichment mode of operation. Such a mode of operation of an enrichment process provides significant flexibility in that it has no bias with respect to what attributes define a CTC; thereby allowing the researcher or clinician to use any maker they choose to define whether the final, enrich product contains CTCs or other cell type relevant to the specific question (i.e., does the CTC have predominately epithelial or mesenchymal characteristics?). Biotechnol. Bioeng. 2009;102: 521–534. © 2008 Wiley Periodicals, Inc.     

Cuprous oxidase activity of yeast Fet3p and human ceruloplasmin: implication for function

The Fet3 protein in Saccharomyces cerevisiae and mammalian ceruloplasmin are multicopper oxidases (MCO) that are required for iron homeostasis via their catalysis of the ferroxidase reaction, 4Fe(2+)+O(2)+4H(+)-->4Fe(3+)+2H(2)O. The enzymes may play an essential role in copper homeostasis since they exhibit a strikingly similar kinetic activity towards Cu(1+) as substrate. In contrast, laccase, an MCO that exhibits weak activity towards Fe(2+), exhibits a similarly weak activity towards Cu(1+). Kinetic analyses of the Fet3p reaction demonstrate that the ferroxidase and cuprous oxidase activities are due to the same electron transfer site on the enzyme. These two ferroxidases are fully competent kinetically to play a major role in maintaining the cuprous-cupric redox balance in aerobic organisms.     

Copper subtype of Alzheimer's disease (AD): Meta-analyses,genetic studies and predictive value of non-ceruloplasmim copper in mild cognitive impairment conversion to full AD

Alzheimer's disease (AD) is the most common form of dementia. A myriad of complex factors contribute to AD, promoting the deposition in plaques of amyloid-beta (Aβ), which is the main constituent of this pathognomonic sign of AD at autopsy brain inspection. Aβ toxicity is related to oxidative stress, which results in synaptic loss in specific brain areas, eventually leading to cognitive decline. Metal, and especially copper, dyshomeostasis is a key factor in these processes. Recent studies have demonstrated that the serum fraction of copper that is not bound to ceruloplasmin (Non-Cp copper, also known as ‘free’ or labile copper) increases in a percentage of AD patients and mild cognitive impairment (MCI) subjects; this is considered a precursor of AD. Non-Cp copper is the exchangeable fraction of low molecular weight copper in serum. It is distinguished from the copper structurally bound to the ceruloplasmin protein, a master protein of iron metabolism. Non-Cp copper levels are higher than normal reference values (range 0–1.6 μmol/L) in about 50% of amnestic MCI subjects and 60% of AD patients, typifying them in a subset of AD. Meta-analyses, genetic studies and a prognostic study evaluating the predictive value of Non-Cp copper in MCI conversion to full AD demonstrate the existence of this copper phenotype of AD.     

In-vitro evaluation of copper/copper oxide nanoparticles cytotoxicity and genotoxicity in normal and cancer lung cell lines

BackgroundNanotoxicology is a major field of study that reveals hazard effects of nanomaterials on the living cells.MethodsIn the present study, Copper/Copper oxide nanoparticles (Cu/CuO NPs) were prepared by the chemical reduction method and characterized by different techniques such as: X-Ray Diffraction, Transmission and Scanning Electron Microscopy. Evaluation of the toxicity of Cu/CuO NPs was performed on 2 types of cells: human lung normal cell lines (WI-38) and human lung carcinoma cell (A549). To assess the toxicity of the prepared Cu/CuOs NPs, the two cell types were exposed to Cu/CuO NPs for 72 h. The half-maximal inhibitory concentration IC₅₀ of Cu/CuO NPs for both cell types was separately determined and used to examine the cell genotoxicity concurrently with the determination of some oxidative stress parameters: nitric oxide, glutathione reduced, hydrogen peroxide, malondialdehyde and superoxide dismutase.ResultsCu/CuO NPs suppressed proliferation and viability of normal and carcinoma lung cells. Treatment of both cell types with their IC₅₀’s of Cu/CuO NPs resulted in DNA damage besides the generation of reactive oxygen species and consequently the generation of a state of oxidative stress.ConclusionOverall, it can be concluded that the IC₅₀'s of the prepared Cu/CuO NPs were cytotoxic and genotoxic to both normal and cancerous lung cells.     

Embryotoxicity of silver ions is diminished by ceruloplasmin—further evidence for its role in the transport of copper

The effect of alimentary administration of silver salts upon embryogenesis in rats was studied. Feeding of female rats throughout the term on a regular diet supplemented with AgCl did not cause alterations of their physiological functions, despite the fact that enzymatically active copper-containing ceruloplasmin (CP) was eliminated from the blood plasma. However, developmental abnormalities of embryos, their prenatal death or the 100% mortality of the newborns in the first 24 h of life was seen. Copper content in placenta and fetal tissues was strongly diminished. Cu, Zn-superoxide dismutase (SOD) activity decreased in cytoplasm of embryonic cells along with a drop, though less pronounced, in the tissues of the pregnant females. Embryotoxicity of AgCl was seriously diminished by repetitive injections of native CP to the pregnant rats. Such treatment resulted in an increase of SOD activity in placenta and embryonic tissues. The mortality of the newborns also became less. It is suggested that the embryotoxic effect of AgCl is caused by its ability to interfere with copper metabolism, in particular by altering the copper-transporting function of CP.     

Kinetics and DFT studies on the reaction of copper(II) complexes and H2O2

Copper(II) complexes supported by bulky tridentate ligands L1^H (N,N-bis(2-quinolylmethyl)-2-phenylethylamine) and L1^Ph (N,N-bis(2-quinolylmethyl)-2,2-diphenylethylamine) have been prepared and their crystal structures as well as some physicochemical properties have been explored. Each complex exhibits a square pyramidal structure containing a coordinated solvent molecule at an equatorial position and a weakly coordinated counter anion (or water) at an axial position. The copper(II) complexes reacted readily with H₂O₂ at a low temperature to give mononuclear hydroperoxo copper(II) complexes. Kinetics and DFT studies have suggested that, in the initial stage of the reaction, deprotonated hydrogen peroxide attacks the cupric ion, presumably at the axial position, to give a hydroperoxo copper(II) complex retaining the coordinated solvent molecule (H ^R ·S). H ^R ·S then loses the solvent to give a tetragonal copper(II)-hydroperoxo complex (H ^R ), in which the –OOH group may occupy an equatorial position. The copper(II)–hydroperoxo complex H ^R exhibits a relatively high O–O bond stretching vibration at 900 cm⁻¹ compared to other previously reported examples.Electronic Supplementary Material Supplementary material is available for this article at     

Effects of Dietary Copper (II) Sulfate and Copper Proteinate on Performance and Blood Indexes of Copper Status in Growing Pigs

160 crossbred (Duroc × Landrace ×Yorkshire) gilts averaged 21.25 kg body weight were used to study the effects of dietary copper (II) sulfate (CuSO₄) and copper proteinate (Cu-Pr) on growth performance, plasma Cu concentration, ceruloplasmin activity, and erythrocyte Cu/Zn-superoxide dismutase (SOD) activity. All pigs were allotted to four treatments and fed with basal diets supplemented with 0 (control), 250 mg /kg Cu as CuSO₄, and 50 and 100 mg/kg Cu as Cu-Pr. Growth performance was determined based on two growth phase (phase 1: days 0 to 15, phase 2: days 15 to 30). After 30 days of the treatment, 16 pig blood samples (four per treatment) were collected for indexes of copper status determination. The experimental results showed that compared with control, pigs fed with 250 mg Cu/kg as CuSO₄ and 100 mg Cu/kg as Cu-Pr had higher average daily gain and average daily feed intake in the whole growth phase (d 0 to 30). In addition, 250 mg Cu/kg as CuSO₄ and 100 mg/kg Cu as Cu-Pr enhanced plasma ceruloplasmin activity (P < 0.05), and 100 mg/kg Cu as Cu-Pr increased erythrocyte Cu/Zn-SOD activity (P < 0.01) compared with the control. There was no obvious treatment response on plasma Cu concentration in the present study.     

Teeth-derived stem cells: A source for cell therapy

Cell therapy is one of the important therapeutic approaches in the treatment of many diseases such as cancer, degenerative diseases, and cardiovascular diseases. Among various cell types, which could be used as cell therapies, stem cell therapy has emerged as powerful tools in the treatment of several diseases. Multipotent stem cells are one of the main classes of stem cells that could originate from different parts of the body such as bone marrow, adipose, placenta, and tooth. Among several types of multipotent stem cells, tooth-derived stem cells (TDSCs) are associated with special properties such as accessible, easy isolation, and low invasive, which have introduced them as a good source for using in the treatment of several diseases such as neural injuries, liver fibrosis, and Cohrn’s disease. Here, we provided an overview of TDSCs particular stem cells from human exfoliated deciduous teeth and clinical application of them. Moreover, we highlighted molecular mechanisms involved in the regulation of dental stem cells fate.     

Effect of medium copper concentration on the growth,uptake and intracellular balance of copper and zinc in Menkes' and normal control cells

The precise nature of the variation in cellular copper load against medium copper concentration is defined using a comprehensive logarithmically incremented series of medium copper concentrations ranging from low levels (4.8 p.p.b.) through normal to toxic levels (40 p.p.m.) in which fibroblasts were grown followed by determination of intracellular content. Menkes' fibroblasts showed an unexpected plateau region of stable intracellular copper content against a change in medium concentration of over 100-fold, albeit only when sufficient copper was present in the medium (0.08–8.0 p.p.m.). Thus, Menkes' cells are clearly capable of balancing uptake/efflux providing copper availability allows. Simultaneous analysis of cellular copper and zinc load at various medium copper concentrations shows an indistinguishable intracellular copper:zinc ratio between the two cell lines. The nature of non-labeled copper uptake by fibroblasts over a 40 min and 7 day period is reported. During the 40 min period copper uptake (20 p.p.m.) was essentially the same in both cell lines. However, copper absorbed was superimposed upon large pre-existing copper pools in the case of Menkes' cells only. Advantages of techniques determining non-labeled copper in copper uptake/efflux experiments are discussed in the light of these results. Fibroblast growth studies showed that, compared with normal cells, Menkes' cells are significantly (P < 0.01) more growth sensitive to extended exposure to low copper concentrations. Thus, Menkes' disease appears to be not only a result of copper maldistribution but also a direct result of an inability of Menkes' cells to function normally in low copper environments.     

The use of trans-4-cotininecarboxylate to construct a polymeric copper(II) azido complex: Hydro(solvo)thermal synthesis, structure and magnetic properties

A new pyridyl-carboxylate ligand, the anion of trans-4-cotininecarboxylic acid, HL, 1, has been used to prepare a new polymeric copper(II) complex, [CuLN₃]_2n, 2, based on a [CuLN₃]₂ dimeric building block. The single crystal structures of both 1 and 2 have been determined and 1 has been found to be in its zwitterionic configuration. The structure of 2 is a one-dimensional tape-like polymeric structure based on an end-on azido-bridged binuclear [Cu₂N₃]₂ backbone moiety. Magnetic studies reveal that 2 is close to paramagnetic from 2 to 300 K with a Curie constant of 1.094 emu K/mol, a Weiss temperature of 0.73 K and a corresponding μ_eff of 2.09 μ_B. A fit of χ_MT for 2 with S₁ = S₂ = ½, yields g = 2.441(6), J = −0.49(3) cm⁻¹, zJ = −0.38(2) cm⁻¹ and N(α) = 0.00053(12) emu/mol, a fit that indicates the presence of both very weak intramolecular intrachain antiferromagnetic exchange coupling within the one-dimensional tape-like chains and very weak interchain antiferromagnetic exchange coupling between these chains.     

Isolation of a copper complex and its rate of appearance in roots of Mimulus guttatus

A Cu-complex was isolated from the roots of copper-tolerant Mimulus guttatus. The elution volume of the complex determined by gel permeation chromatography was similar to that of rat-liver cadmium thionein. The complex was heat stable, had a relatively high ratio of absorbance at 254 nm: 280 nm and incorporated ³⁵S. The complex, purified using a combination of gel permeation chromatography and anion-exchange chromatography, contained more glutamine/glutamic acid and glycine residues than mammalian metallothioneins. The amount of the complex in roots increased after 5 h growth in a solution containing 16 M Cu. Induction was preceded by an increase in the concentrations in root tissue of unknown compounds containing sulphur which may serve as precursors. The availability of these compounds appeared to regulate the rate of synthesis of this Cu-complex.Abbreviations CuBP copper-binding protein - HPLC highperformance liquid chromatography - MT metallothionein - Th thionein - Tris 2-amino-2-(hydroxymethyl)1-3-propanediol     

Intracellular transport and degradation of 125I-labelled denatured serum albumin in isolated nonparenchymal rat liver cells

The intracellular movement, following uptake of ¹²⁵I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker β-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker β-acetylglucosaminidase, suggesting lysosomal degradation.     

Quenching of bathocuproine disulfonate fluorescence by Cu(I) as a basis for copper quantification

In this paper we report the up to now ignored fluorescence properties of the specific Cu(I)-chelator bathocuproine disulfonate and their application in assays of total copper and Cu(I). The method is based on the linear quenching of the bathocuproine disulfonate emission at 770 nm (lambda(ex)580 nm) by increasing concentrations of Cu(I), at pH 7.5. Copper concentrations as low as 0.1 microM can be determined. Other metal ions (iron, manganese, zinc, cadmium, cobalt, nickel) do not interfere. The procedure for total copper determination in proteins includes HCl treatment to release the copper, neutralization to pH 7.5 in the presence of citrate to stabilize the copper, and reduction of the copper to Cu(I) by ascorbate in the presence of the chelator. This assay gave results coincident with the analysis by atomic absorption spectroscopy in two selected proteins. In addition, conditions are described (omitting HCl treatment and reduction by ascorbate) for direct measurement of Cu(I) in native proteins, as illustrated for the Escherichia coli NADH dehydrogenase-2. Data show that the fluorometric assays described in this paper are simple and convenient procedures for total copper and direct Cu(I) quantification in determined biological samples.