Protein-bound Copper Research Articles

Time course of gold-induced accumulation of copper and zinc and the effects of dimercaptosuccinate and cadmium on gold metabolism in rat kidney

After the s.c. administration of sodium aurothiomalate (SATM, 10 mg Au/kg) to male rats, the gold content of the kidney increased to a maximum after 4 days and thereafter declined slowly. The total copper content of the kidney increased at least until 11 days after SATM treatment but was not simply a function of the renal gold content. Gold and copper accumulated in the metallothionein-like, low molecular weight protein fraction and the initial uptake of gold by this fraction appeared to be related to the accumulation of copper. The Zn 2+ content of the kidney was initially unchanged but later increased with the additional Zn 2+ bound predominantly to the non-soluble components. The accumulation of low molecular weight protein-bound copper was not related either to the Zn 2+ content of this fraction or to the total Zn 2+ content of the kidney. Daily administration of dimercaptosuccinic acid (DMSA, 50 mg/kg, i.p.) for 2 weeks to SATM-pretreated rats resulted in a 50% reduction in the concentration of gold in the non-soluble fraction and in both the high and low molecular weight protein components of the soluble fraction. Copper, which accumulated in the non-soluble components and in the soluble low molecular weight protein, was lost only from the non-soluble components. Uptake of gold by the kidneys was increased when rats were pretreated with Cd 2+. The accumulation of gold in the low molecular weight protein fraction was also increased, but most of the additional gold in the kidneys was accumulated by the non-soluble components. SATM had no effect on the renal concentration or subcellular distribution of Cd 2+. It was concluded that the soluble low molecular weight metal binding protein does not have prominent regulatory or protective functions in the renal metabolism of gold.

Use of direct current argon plasma as a detector in gel filtration chromatography of biological fluids

A direct-current argon plasma spectrometer has been interfaced with a gel filtration chromatography column to serve as a multi-element-specific detector. This analytical system was used to speciate protein-bound copper, iron, and zinc in serum and intravenous infusion fluids. The operating parameters of the direct current argon plasma including instrumental drift, detection limits, effect of background levels on the calibration graphs. and accuracy were optimized. Calibrations had to be repeated every hour to compensate for instrumental drift. The detection limits of this system (3.2, 3.9 and 9.3 μg l −1 for copper, iron and zinc, respectively) are adequate for the determination of most species containing those elements in the column effluent.

16] Protein determination in membrane and lipoprotein samples: Manual and automated procedures

Publisher Summary This chapter describes the protein determination in membrane and lipoprotein samples. A variety of methods have proved to be effective in estimating the protein content of water-soluble samples. The procedure used is based on this modified Lowry method. It is found that by adding sodium dodecyl sulfate to the alkali reagent, samples can be assayed directly without prior solubilization or delipidation. An increase in the copper tartrate concentration facilitates quantitation of protein in the presence of sucrose and EDTA. Color formation depends mainly on reduction of the Folin-Ciocalteu reagent by protein-bound copper and proceeds in two distinct steps. The wavelength used routinely in the modified Lowry procedure was chosen as a compromise between increased absorption of the final blue reduction product with longer wavelengths and the practical limitations of most spectrophotometers. The absorption peak of the blue chromophore extends through much of the visible spectrum in a broad plateau and reaches maximum at 750 nm. The effectiveness and rapidity of this modified Lowry procedure as compared to the original Lowry procedure for assaying complex biological systems are also discussed.

Separation of protein-bound copper and zinc in human plasma by means of gel filtration -ion-exchange chromatography.

A procedure for the separation of protein-bound copper and zinc fractions and of potassium and magnesium in plasma, involving chromatography on columns of DEAE-Sepharose CL-6B, is described. Copper, magnesium and zinc were measured by atomic-absorption spectroscopy and potassium by flame photometry. Most plasma samples yielded one copper and two zinc fractions and these were identified by the use of protein “markers.” Most of the copper appeared to be bound to caeruloplasmin and a lesser amount to albumin, whereas zinc appeared to be bound mainly to globulins with a smaller, variable, amount bound to albumin. Magnesium and potassium appeared as single peaks eluting in approximately the same fractions, ahead of the albumin peak.

The formation and nature of the mixed valence copper- d-penicillamine-chloride cluster in aqueous solution and its relevance to the treatment of Wilson's disease

Complex formation between d-penicillamine (Pen) and copper(II) ions has been studied under simulated physiological conditions in both the presence and absence of the blood plasma constituents albumin, alanine, histidine, and zinc(II). Chromatographic and uv/vis and electron spin resonance (esr) spectroscopic methods were used. The major species formed, at neutral pH and 0.15 mol dm -3 NaCl, is the violet species which is shown to have the same stoichiometry as the recently reported solid-state complex, i.e., [Cu 8 I Cu 6 II (Pen) 12 Cl] 5-. The rate of formation of this species (MVC) is shown to be dependent on the Cu concentration, Cu:Pen ratio, relative Cl - ion concentration, pH, and temperature. Formation is inhibited by the presence of O 2 and biological chelates. At the concentration levels found in blood plasma it is unlikely that the MVC ion has any significance in the therapeutic action of penicillamine in the treatment of Wilson's disease. Reexamination of the aqueous Cu-albumin-pen system reinforces earlier findings that pen is unable to mobilize Cu that is bound to albumin. Significant binding of pen to the protein is observed but is not related to any protein-bound copper ions. Evidence that ternary complexes of the type amino acid-Cu-Pen can form in blood plasma is presented. These are unlikely, however, to be physiologically significant and the copper depletion induced by Pen in Wilson's disease cases must be elsewhere than in the blood plasma compartment.

Detection of copper on polyacrylamide gels

A color test for the localization of copper on polyacrylamide gels is described. The test is based upon the quenching of fluorescence of bathocuproine sulfonate by Cu 1+ and is sensitive to 0.1 nmol of free or protein-bound copper. There is no false positive reaction with 10 nmol of hemeprotein, free Fe 3+, Fe 2+, Co 2+, or Mn 2+.

Variable sensitivity in the microbiuret assay of protein

Microbiuret methods have been introduced which have sensitivity similar to that of the method of Lowry et al. (1) and are claimed to be less subject to interference (2–5). Each method has three steps: (I) formation of the protein-copper biuret complex in alkaline solution, (II) separation of excess copper reagent from protein-bound copper, and (III) colorimetric assay of the latter with diethyldithiocarbamate. They differ chiefly in steps I and II. However, the concentration of dry bovine serum albumin (BSA) reported to give absorbance 1.0 in these methods ranges from 64 to 99 μg/ml of final coloured solution. In preliminary studies in this laboratory these variations were confirmed and in one case [the method of Westley and Lambeth (3)] linearity and sensitivity were significantly improved by addition of detergents in step III. This was surprising since albumin is added in step III of that procedure to prevent precipitation of the poorly soluble copper complex. This report shows that absorbance and stability of the copper-diethyldithiocarbamate complex is markedly affected both by the concentration of alkali and by the presence or absence of detergents. The method of Klungsöyr (4), in which excess alkaline copper tartrate reagent is removed by adsorption on a Sephadex column, is probably the simplest for multiple assays and a modified procedure giving a reproducible high sensitivity of absorbance 1.0 for 51–53 μg of dry BSA/ml of colour is described.

Determination of protein-bound copper and zinc in some organs of the cuttlefish Sepia officinalis L.

1. 1. The copper, zinc and iron content of blood and water soluble extracts from several organs of Sepia officinalis L. have been determined with atomic absorption spectrometry. 2. 2. The relatively high concentration of copper and zinc in the branchial gland points to its probable role in trace element metabolism. 3. 3. After gel filtration a low molecular weight copper and zinc containing fraction can be isolated from the gland. 4. 4. A possible role of this fraction in hemocyanin biosynthesis is postulated.

Histidine as an essential residue in the active site of the copper enzyme galactose oxidase

The pH dependence of the oxidation of β-methyl- d-galactopyranoside by galactose oxidase at 1.33 m m O 2 has been determined. The k cat exhibits a bell-shaped dependence on the ionization of at least two groups in the enzyme-substrate complex, p K b ' = 6.3 and p K a ' = 7.1, respectively. The pH-independent value for k cat at 1.33 m m O 2 (nonsaturating) and saturating glycoside is 1435 s −; the pH optimum is 6.7. Galactose oxidase is inactivated rapidly by iodoacetamide. Although the reaction is much slower, iodoacetate also inactivates the enzyme. The inactivation by iodoacetamide obeys saturation kinetics; at pH 7.0 k 3 = 2.19 min −1 and K i = 5.1 mM; k 3 but not K i exhibits a bell-shaped pH dependence, with p K a values of 6.3 and 7.6, respectively. Labeling with [ 14C]iodoacetamide establishes that one carboxamidomethyl group is incorporated per enzyme molecule. This incorporation parallels the loss of enzymatic activity. Only N-3-carboxymethylhistidine is detected in chromatograms following hydrolysis of the labeled protein. The protein-bound copper is not lost as a consequence of alkylation. Apogalactose oxidase does not react with iodoacetamide. The alkylation is inhibited by the oxidation of an active center tryptophan residue (s) by N-bromosuccinimide. The fraction of residual enzyme activity remaining after tryptophan oxidation corresponds to the extent of labeling by [ 14C]iodoacetamide. Although alkylation causes little change in the spin Hamiltonian parameters of the Cu(II) atom, it nearly abolishes both the optical activity and optical absorbance of the metal. The native tryptophan fluorescence of the enzyme, which is a sensitive probe of its active site, is also markedly affected. Since binding of a substrate, β-methyl- d-galactopyranoside, reduces fluorescence as it does in the active enzyme and binding of CN − at the Cu(II) site as detected by electron spin resonance appears unaffected by the alkylation, the effect of alkylation is on catalysis, per se. Both a catalytic and a subtle conformational role for the active site histidine are inferred from the results.

Combined gel filtration, biuret/copper method compared with an immunochemical method for urinary protein measurement.

We compared an immunochemical method specific for plasma proteins with a chemical method, in which interfering substances are separated by gel filtration and "total" urinary protein is determined by the biuret reaction followed by reaction of protein-bound copper with diethyldithiocarbamate after a second gel filtration to remove nonprotein-bound copper. More than 250 24-h urine samples were analyzed by each method. There was linear agreement and a correlation of 0.96 between the two methods, but urinary protein values determined by the immunochemical method, especially for patients with multiple myeloma, were lower than by the chemical method.

Blockade by metal complexing agents and by catalase of the effects of arachidonic acid on platelets: Relevance to the study of anti-inflammatory mechanisms

Metal-chelating agents inhibited platelet aggregation and the accompanying generation of rabbit aorta contracting and PG-like activities, when platelets were challenged with arachidonic acid. Inhibition required the presence of the chelating agents in the medium, and was insured by reagents avid for free or protein-bound copper. Catalase also prevented aggregation and generation of pharmacologically active substances; its activity was reversed by aminothiol agents and by Cu 2+ and Zn 2+, shown previously to potentiate the platelet effects of arachidonic acid. Inhibition by indomethacin was not prevented by amino-thiol drugs nor by Cu 2+ or Zn 2+. The catalase-induced inhibition was not affected by scavenging of thiol groups; this rules out, as a mechanism of action of catalase, the increased destruction of lipoperoxides by glutathione peroxidase, which requires reduced glutathione as hydrogen donor. The results are compatible with the hypothesis that the agent that mediates platelet aggregation by arachidonic acid is a lipoperoxide, requiring the presence either of H 2O 2 or of a similarly catalase-sensitive substance to be generated.

Effect of metal-complexing agents on the oxygenase activity of sheep vesicular glands

A variety of agents that are known to complex copper can reversibly inhibit the oxygenation of unsaturated fatty acids by particulate preparations from sheep vesicular glands. The time-dependent activation of the oxygenase preparation by phenol, on the other hand, was not affected by the copper chelator, diethyldithiocarbamate. The results suggest that protein-bound copper could play a role in the interaction of the oxygenase enzyme with oxygen and the fatty acid substrates. Other findings indicate that phenanthroline type compounds may inhibit both the oxygenation reaction and phenol activation by binding to an hydrophobic site on the oxygenase.

Radiation Chemical Studies of Hemocyanin in Oxygen-Free Media

The oxygen-carrying capacity of Busycon and Limulus hemocyanins is eliminated following irradiation of the protein with cobalt-60 γ-rays in neutral, oxygen-free media. The loss of oxygen-carrying capacity of the protein is measured from the change of the characteristic copper band at 340 nm, before and after irradiation. The limiting ${\rm G}(-{\rm O}_{2})$ values in the absence of scavengers are 0.026 and 0.044 for Busycon and Limulus hemocyanins, respectively. Radiation effects in oxygen-free media on Busycon and Limulus hemocyanins can either be enhanced or prevented by appropriate scavengers. The Busycon methemocyanin activity is restored by the hydrated electron, $e_{{\rm aq}}^{-}$, and the secondary ${\rm CO}_{2}{}^{-}$ radical. The loss of oxygen-carrying capacity of hemocyanin by γ irradiation is attributed, therefore, to the OH radical which oxidizes the protein-bound copper at the active site, thus forming methemocyanin. The restoration of the oxygen-carrying capacity of Busycon methemocyanin is...

An X-ray Crystallographic Study of Ceruloplasmin: Determination of Molecular Weight

Human ceruloplasmin crystallizes in the tetragonal space group I4 with a = b = 268 A and c = 129 A. The asymmetric unit consists of two molecules of ceruloplasmin of molecular weight 132,000. Taken in conjunction with the published value for the total protein-bound copper, these results indicate that there are 6 copper atoms in each ceruloplasmin molecule.

Quantitative estimation of protein: Separation of alkaline protein-copper complex from excess copper on Sephadex G-25

When a reaction mixture containing protein and divalent copper in an alkaline tartrate solution was passed through a column of Sephadex G-25, the protein-copper complex was excluded as an entity, while the copper in the tartrate complex was taken up by the gel by what appears to be a specific binding process. This was used as a method for quantitative estimation of protein: samples were treated with a modified “biuret” reagent and passed through columns of G-25, and protein-bound copper was determined colorimetrically in the basic eluate after addition of sodium diethyldithiocarbamate. The procedure increased the sensitivity of the biuret reaction 50-fold, and decreased interferences of several substances commonly encountered.

The identification of metal-protein complexes by gel chromatography and neutron activation analysis

A method for the identification of protein-metal complexes in biological fluids by a combination of gel chromatography and neutron activation analysis is proposed. The distribution of protein-bound copper in human serum is studied as an example of the method.

Ceruloplasmin-Anion Interactions: INDUCED SPECTRAL TRANSITIONS IN THE VISIBLE RANGE

Changes in the visible spectrum of human ceruloplasmin resulting from the interaction of the protein-copper chromophore with azide, cyanide, thiocyanate, and cyanate have been examined. Absorption maxima determined for the various protein-anion complexes were the following: N3−, 375 mµ with a shoulder at 435 mµ; CN−, 378 mµ; NCS−, 375 mµ and 435 mµ; NCO−, 395 mµ. The spectral transitions induced by N3−, NCS−, and NCO− produced reductions in the absorption of the protein-copper chromophore of 75, 62, and 65%, respectively. Complex formation in the case of azide and cyanate was reversible, as indicated by a quantitative recovery of both chromophore absorption (610 mµ) and oxidase activity upon removal of the anion. Ceruloplasmin regenerated from the thiocyanate complex exhibited a 10% reduction in these two properties. Protein-bound copper was not removed by either azide, thiocyanate, or cyanate treatment. In the presence of 0.01 M CN− at pH 5.5, ceruloplasmin rapidly lost 1 g atom of copper per 160,000 g of protein. This resulted in the permanent loss of approximately one-third of the absorption at 610 mµ and 54% of the initial oxidase activity. Additional copper was slowly lost upon long term incubation with cyanide. The results of this investigation indicate that predominantly 1 chromophoric cupric copper atom interacts in a reversible manner with N3−, NCS−, and NCO− under the conditions studied. Furthermore, this cupric atom appears to be located on the surface of the molecule exposed to the external environment of the protein and is probably the copper atom readily removed by cyanide.

Inhibition of dopamine beta-hydroxylase by aquayamycin.

Aquayamycin was found to be one of the most potent inhibitors of dopamine β-hydroxylase. Aquayamycin at 4×10-7 M inhibited the enzyme by 50% with a Ki value of 2.1×10-7 M. The inhibitionwas non-competitive with substrate and was not affected by cof actors, i.e. ascorbic acid or fumarate. The inhibitory mechanism is possibly due to chelating action of aquayamycin on protein-bound copper. However, the addition of Cu++ did not reverse the inhibition.

Radiation of hemocyanin: inactivation and reactivation of oxygen-carrying capacity.

Oxygen-carrying capacity of hemocyanin from Limulus and Busycotypus (Busycon) decreases with increasing radiation, giving initial yield values for G(-O(2),) of 1.1 and 1.0, respectively. High radiation doses regenerate this capacity of Busycotypus hemocyanin. These effects are attributed largely to the dual nature of hydrogen peroxide, which, at low concentrations, oxidizes protein-bound copper and at high concentrations, that is, at high doses, reduces oxidized copper. The ability of hemocyanin to decompose hydrogen peroxide is relatively unaffected by irradiation, which suggests that copper atoms at the active sites are not all equivalent. The catalase-like activity of Busycotypus hemocyanin can be simulated by amino acid chelates of copper, including arginine, histidine, and glycine.

An Electron Paramagnetic Resonance Study of 3,4-Dihydroxyphenylethylamine β-Hydroxylase

Abstract 1. The role of the copper in highly purified bovine adrenal 3,4-dihydroxyphenylethylamine β-hydroxylase (dopamine β-hydroxylase) has been studied with the use of electron paramagnetic resonance techniques. The results confirm our previous findings. Most of the protein-bound Cu2+ is reduced by treatment of the enzyme with ascorbate; a large part of the Cu+ in the reduced enzyme is reoxidized during the hydroxylation of substrate. These results support a reduction-oxidation role for the protein-bound copper. 2. Fumarate, an activator of dopamine β-hydroxylase, appears to favor the oxidation of Cu+ to Cu2+. This effect of fumarate is discussed in terms of the mechanism of action of dopamine β-hydroxylase.