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 K_D 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.     

Metal components analysis of metallothionein-III in the brain sections of metallothionein-I and metallothionein-II null mice exposed to mercury vapor with HPLC/ICP-MS

Mercury vapor is effectively absorbed via inhalation and easily passes through the blood–brain barrier; therefore, mercury poisoning with primarily central nervous system symptoms occurs. Metallothionein (MT) is a cysteine-rich metal-binding protein and plays a protective role in heavy-metal poisoning and it is associated with the metabolism of trace elements. Two MT isoforms, MT-I and MT-II, are expressed coordinately in all mammalian tissues, whereas MT-III is a brain-specific member of the MT family. MT-III binds zinc and copper physiologically and is seemed to have important neurophysiological and neuromodulatory functions. The MT functions and metal components of MTs in the brain after mercury vapor exposure are of much interest; however, until now they have not been fully examined. In this study, the influences of the lack of MT-I and MT-II on mercury accumulation in the brain and the changes of zinc and copper concentrations and metal components of MTs were examined after mercury vapor exposure by using MT-I, II null mice and 129/Sv (wild-type) mice as experimental animals. MT-I, II null mice and wild-type mice were exposed to mercury vapor or an air stream for 2 h and were killed 24 h later. The brain was dissected into the cerebral cortex, the cerebellum, and the hippocampus. The concentrations of mercury in each brain section were determined by cold vapor atomic absorption spectrometry. The concentrations of mercury, copper, and zinc in each brain section were determined by inductively coupled plasma mass spectrometry (ICP-MS). The mercury accumulated in brains after mercury vapor exposure for MT-I, II null mice and wild-type mice. The mercury levels of MT-I, II null mice in each brain section were significantly higher than those of wild-type mice after mercury vapor exposure. A significant change of zinc concentrations with the following mercury vapor exposure for MT-I, II null mice was observed only in the cerebellum analyzed by two-way analysis of variance. As for zinc, the copper concentrations only changed significantly in the cerebellum. Metal components of metal-binding proteins of soluble fractions in the brain sections were analyzed by size-exclusion high-performance liquid chromatography (HPLC) connected with ICP-MS. From the results of HPLC/ICP-MS analyses, it was concluded that the mercury components of MT-III and high molecular weight metal-binding proteins in the cerebellum of MT-I, II null mice were much higher than those of wild-type mice. It was suggested that MT-III is associated with the storage of mercury in conditions lacking MT-I, and MT-II. It was also suggested that the physiological role of MT-III and some kind of high molecular weight proteins might be impaired by exposure to mercury vapor and lack of MT-I and MT-II.     

A Critical Comparison of Hanging Mercury Drop Electrode and Long Lasting Sessile Drop Mercury Electrode in A.C. Anodic Stripping Voltammerty

Abstract

The hanging mercury drop electrode (HMDE) and the long lasting sessile drop mercury electrode (LLSDME) currently used in trace metal analysis are critically compared. Alternating current anodic stripping voltammetry (AC-ASV) and alternating current linear sweep voltammetry (AC-LSV) respectively are employed for the determination of Zn,Cd, Pb, Cu and Fe.

The influence of the electrolysis time, stirring rate, a.c. amplitude, frequency and scan rate on the peak current is evaluated.

Analytical measurements were carried out by the standard addition method, using 0.02 mol/l HClO₄ + 0.1 mol/l NaClO₄ (Zn, Cd, Pb, Cu) and 0.1 mol/l (COONa)₂, (Fe), respectively as supporting electrolytes.     

Metallomic study on plasma samples from Nile tilapia using SR-XRF and GFAAS after separation by 2D PAGE: initial results

An investigation was made on plasma samples obtained after protein separation. The proteome of the plasma of Nile tilapia (Oreochromis niloticus) was separated by 2D PAGE, and manganese and zinc in protein spots was qualitatively and quantitatively determined by synchrotron radiation X-ray fluorescence (SR-XRF) and graphite furnace atomic absorption spectrometry (GFAAS). Manganese and zinc are present in four and six plasma protein spots, respectively. These ions are bound to proteins with molecular weights ranging from 19 to 70?kDa and with isoelectric point (pI) ranging from 4.7 to 6.3. The concentrations of manganese and zinc bound to these proteins as determined by GFAAS following acid digestion of the spots range from 0.8 to 2.6?mg of manganese, and from 1.0 to 6.3?mg of zinc, respectively, per g of protein.

Purification and Characterization of Superoxide Dismutase （SOD） from Camellia Pollen

A superoxide dismutase（ SOD ） was purified to homogeneity from fresh camellia pollen by means of ammonium sulfate precipitation and column chromatography with DEAE-cellulose（ DE52 ）, Sephadex G-100 and phenyl sepharose^TM 6 Fast Flow columns. Its specific activity could reach to 4034 U/mg protein and it was determined to be Cu/ Zn-SOD according to its different sensitivities to different inhibitors. The molecular weight of the SOD and its subunit were 69500 and 34700, respectively, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS- PAGE）, which implicates that the SOD in camellia pollen is a dimmer composed of two identical subunits. The isoelectric point of the enzyme was determined to be 4. 1 by isoelectric focusing electrophoresis and the N-terminal amino acid was identified to be Gly by the DNS-Cl method. Its α-Helix was also calculated to be approximately 21.8% according to the circular dichroism（CD） spectra.     

Purification and Properties of Superoxide Dismutase（SOD） in Allium Sativum

Superoxide dismutases(SODs) were purified to homogeneity from Allium Sativum by means of ammoni-um sulfate precipitation and column chromatography with DEAE--cellulose (DE52) and Sephadex G-75. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-AGE), Allium Sativum is predicted to contain four SODs. The molecular weights of the native SODs are 41.3 kD, 37. 0 kD, 35.2 kD and 31.0 kD, which consist of subunits of 20. 7 kD, 18. 4 kD, 17. 7 kD and 15.4 kD respectively. Because of their specific sensitivity to hydrogen peroxide, cyanogens potassium and chloroform-alcohol, the SODs in Allium Sativum appear to be Cu, Zn-SOD isoenzymes. The isoelectric analysis indicates that three of the four isoermymes are acidic proteins with isoelectric points at pH 3.5, 3.7 and 4. 0, respectively, and the fourth one is a basic pro-tein with isoeletric point at pH 8. 5.     

A family of high-buffering capacity diamino sulfate isoelectric buffers for pH-biased isoelectric trapping separations

pH-biased isoelectric trapping separations are hindered by the lack of suitable isoelectric buffers with pI values in the 5.8 < pI range. Two generic approaches are described here for the cost-effective synthesis of a family of diamino sulfate buffers that have high buffering capacities in their isoelectric state: the first approach relies on the sulfation of existing, commercially available diamino alcohol intermediates, the second approach calls for the synthesis of diamino alcohols from epichlorohydrin and widely available secondary amines, and subsequent sulfation of the new diamino alcohol. The diamino sulfate buffers are recovered in isoelectric state, in high purity. Four members of the family having pI values in the 5.8 < pI < 8.9 range have been synthesized, analytically characterized by capillary electrophoresis (CE), electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS), 1-D and 2-D nuclear magnetic resonance (NMR) spectroscopy, and X-ray crystallography. All four diamino sulfates have been successfully used as pH biasers in the receiving stream in preparative-scale pH-biased isoelectric trapping protein separations.     

Capillary electrophoresis of peptides and proteins in isoelectric buffers: an update

Capillary electrophoresis in acidic, isoelectric buffers is a novel methodology allowing fast protein and peptide analysis in uncoated capillaries. Due to the low pH adopted and to the use of dynamic coating with cellulose derivatives, silanol ionization is essentially suppressed and little interaction of macromolecules with the untreated wall occurs. In addition, due to the low conductivity of quasi-stationary, isoelectric buffers, high-voltage gradients can be applied (up to 800 V/cm) permitting fast peptide analysis with a high resolving power due to minimal diffusional peak spreading. Four such buffers are here described: cysteic acid (Cys-A, pI 1.85), iminodiacetic acid (IDA, pI 2.23), aspartic acid (Asp, pI 2.77) and glutamic acid (Glu, pI 3.22). A number of applications are reported, ranging from food analysis to the study of folding/unfolding transitions of proteins.     

Purification and Characterization of a Cu,Zn-SOD from Garlic (Allium sativum L.). Antioxidant Effect on Tumoral Cell Lines

Crude garlic extract contains one Mn-superoxide dismutase designated as SOD1 and two Cu,Zn superoxide dismutases as SOD2 and SOD3. The major isoform SOD2 was purified to homogeneity by Sephacryl S200-HR gel filtration, DEAE Sepharose ion exchange chromatography, and chromatofocusing using PBE 94. SOD2 was purified 82-fold with a specific activity of 4,960 U/mg protein. This enzyme was stable in a broad pH range from 5.0 to 10.0 and at various temperatures from 25 to 60°C. The native molecular mass of SOD2 estimated by high performance liquid chromatography on TSK gel G2000SW column was 39 kDa. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis showed a single band near 18 kDa, suggesting that native enzyme was homodimeric. The isoelectric point as determined by chromatofocusing was 5. Analysis of its N terminal amino acid sequence revealed high sequence homology with several other cytosolic Cu,Zn-SODs from plants. Exposure of cancer cell lines to garlic Cu,Zn-SOD2 led to a significant decrease in superoxide content with a concomitant rise in intracellular peroxides, indicating that the enzyme is active in mammalian cells and could, therefore, be used in pharmacological applications.     

Expression of Cu,Zn-superoxide dismutase gene from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its resistance to oxidative stress

The gene for the Cu, Zn-superoxide dismutase (SOD) from the yeast Saccharomyces cerevisiae was cloned, characterized, and overexpressed in the methylotrophic Pichia pastoris. The sod gene sequence obtained is 465 bp and encodes 154 amino acid residues. The sod gene sequence was cloned into the pPIC9K vector, yielding pAB22. The linearized pAB22 DNA, digested with restriction enzyme SacI, was transformed into the genome of the GS115 strain of the yeast P. pastoris. The SOD was purified from the cultured yeast by ammonium sulfate precipitation and DEAE-cellulose column chromatography. This relatively simple purification method produced a single band on analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The overexpressed SOD protein was shown to have immunologically biologic activity and to be enzymatically active. The yeast overexpressing Cu, Zn-SOD appeared to be more resistant to oxidative stress such as paraquat, menadione, and heat shock.     

Effect of acidic treatment on metal adsorptions of pitch-based activated carbon fibers

In this work, the pitch-based activated carbon fibers (ACFs) were prepared by nitric acid to investigate the multi-metal adsorption in interfacial and textural points of view. N2/77 K adsorption isotherm characteristics, including the specific surface area and micropore volume, were studied by BET specific surface area and t-plot methods, respectively. As a result, the specific surface area of the almost neutral ACFs in nature significantly decreased with nitric acid treatment, probably due to the widening of micropores. However the total acidity, including the carboxyl groups, on carbon surfaces was extremely induced during the acidic surface treatment. From the adsorptions of Cu2+ and Ni2+, it was revealed that the adsorption capacity of metal ions was mainly influenced by the weakly acidic functional groups such as lactones on the carbon surfaces at pH < pI (isoelectric point), and by the strongly acidic functional groups such as carboxyl groups at pH > pI.     

An Electrochemical Flow Cell For The Detection Of Metal Complexes

Abstract

An electrochemical flow cell has been developed using reticulated vitreous carbon (RVC) coated with a thin film of mercury. The flow cell and electrode components are inexpensive and easily constructed. Reproducible results have been obtained for concentrations as low as 100 ppb. Peak currents were dependent on flow rate, electrode size, and mercury deposition time. Oxygen interference was reduced by nitrogen purging to enable analysis of sub-ppm metal concentrations. The cell was tested as a detector for metal complexes eluated from a modified gel permeation chromatographic column.     

New isoelectric buffers for capillary electrophoresis: N-carboxymethylated polyethyleneimine as a macromolecular isoelectric buffer

Isoelectric buffers are attractive for electrophoresis because of their low conductivity, and their compatibility with indirect photometric detection in capillary electrophoresis (CE) where they do not interfere with the detection by exhibiting competitive displacement of the UV-absorbing probe ion. N-carboxymethylated polyethyleneimine (CMPEI) was prepared by introducing a half molar equivalent of carboxylate groups onto a polyethyleneimine backbone. Its isoelectric point determined by conductometric titration and from the pH of its dilute aqueous solution is approx. 6.8, which allows isoelectric buffering at a lower pH compared to histidine (pI7.7). Although the isoelectric point is somewhat diffuse, as expected for a polymeric compound, it exhibits a buffering capacity at a pI point of about twice that of histidine. Studies of electroosmotic flow (EOF) profile at various pH values in fused silica capillaries showed that CMPEI adsorbs onto the fused silica wall and reverses the EOF at pH < 6.5. CMPEI was applied as a buffer in an electrolyte containing 0.5 mM of the anionic dye tartrazine used as the probe for indirect detection of anions. The separation system exhibited a stable baseline, no system peaks, separation efficiencies of up to 195,000 theoretical plates, and detection limits down to 0.2 microM or 2 amol of injected analyte.     

Synthesis,crystal structure,and SOD-like activity of two copper(II) complexes with hydroxymethyl pendants

To mimic the active site of Cu,Zn-SOD, new copper(II) complexes (1, [Cu₂(HL)₂(L)₂](ClO₄)₂; 2, [Cu(HL)₂(phen)](ClO₄)₂; where HL?=?(N-methyl-2-methylol)imidazole, phen?=?1,10-phenanthroline) have been synthesized and characterized by elemental analysis, IR, and single-crystal X-ray diffraction. Copper(II) in 1 is four-coordinate by a N₂O₂ plane with two copper(II) ions bridged with two oxygens from the deprotonated hydroxylmethyl pendants. Each Cu²⁺ in 2 is coordinated by four nitrogens from two HL and one co-ligand of 1,10-phenanthroline. Electrochemistry of the complexes was studied by cyclic voltammetry. The atomic net charges distribution and frontier molecular orbital energies were obtained by Gaussian 98 program with DFT method at B3LYP/lanl2dz level. The SOD-like activity (IC₅₀) of 1 and 2 were measured as 0.10?±?0.01 and 0.19?±?0.01?μM by NBT assay at pH 7.8. The higher SOD activity of 1 could be attributed to the coordination configuration and the labile hydroxymethyl pendants.     

A selective chemosensor for detection of Ag(I) and Cu(I) based on an acenaphthoquinone derivative and its complexes

A new Schiff base, acenaphthoquinone bis(diphenylmethlenehydrazone) (L), was synthesized and employed as a chemosensor for detecting Ag(I) and Cu(I). Experimental results showed that the chemosensor exhibited high selectivity and sensitivity. The sensitivity of the chemosensor for Ag(I) or Cu(I) was not affected by other metal ions, such as Ni(II), Nd(III), Zn(II), Fe(III), Cu(II), Na(I), La(III), K(I), and Co(II). Complexes 1 and 2 were synthesized by coordination of L with Ag(I) and Cu(I), respectively. The crystal structures of 1 and 2 were determined by single-crystal X-ray diffraction. They had the same space group P2₁/c. Based on theoretical calculation, mechanism of the chemosensor detecting Ag(I) and Cu(I) was suggested.     

Synthesis of ditopic ligands for the selective extraction of copper(II) nitrate and crystal structures of their Cu(II) complexes

We have synthesized two ditopic ligands for selective extraction of copper(II) nitrate. We also synthesized one cation-only binding analog for comparison. All three ligands were characterized by conventional techniques. Competitive two-phase metal ion solvent extraction experiments were performed at 25 °C over a period of 24 h. These ligands showed significant selectivity for Cu(II) ions, having the ditopic ligands extract 81 and 73% of the Cu(II) ions in a solution of different metal ions {Ni(II), Co(II), Cu(II), Zn(II), Cd(II), Pb(II)} at pH 5.09. Competitive transport experiments (water/chloroform/water) were undertaken employing each ligand separately as the ionophore in the membrane (chloroform) phase. No metal ion transport was observed, but a large concentration of Cu(II) was present in the membrane phase. Competitive anion extraction and transport were carried out with the ditopic ligands, yielding selective extraction and transport of nitrate. Furthermore, a pH isotherm of the best ditopic ligand (H₂L²) with Cu(II) was determined from pH 1.0 to 6.0, producing a pH_½ value of approximately 2.6. Finally, crystal structures of the ditopic ligands complexed with Cu(II) were determined and refined. The coordination geometry around the metal centers are distorted square planar and the Cu(II)-donor bond lengths fall within the normal range.     

The effect of inorganic particulates on the ASV signals of Cd, Pb and Cu

The potential influence of inorganic particulates on the ASV response of < 100-microg/l. levels of Cd, Pb and Cu, at a thin film mercury electrode, was examined by adding various weights of the solids to the acetate background electrolyte solution. Materials added included the hydrous oxides of Mn(IV), FE(III) or Al(III), clay minerals (kaolinite, illite, montmorillonite) and some contaminated sediments. Abrasion of the mercury film was minimized by deaerating the turbid solutions before their transfer to the measuring cell. The hydrous oxides specifically sorbed all three metal ions, resulting in peak-size changes that varied in magnitude with pH. With the clays, only sorption of Pb by illite or montmorillonite was detected. The presence of the solids had little effect on the peak position or half-peak breadth of the Cd or Pb signals, but the Cu peak parameters changed, indicating some hydroxy-species formation at higher pH. Some contaminated sediment samples released a significant fraction of their total metal content into acetate buffer solutions. Shielding the mercury film with a semipermeable membrane had a similar effect to filtering the suspension before analysis, but diffusion equilibrium was only slowly achieved (> 12 hr).     

Synthesis,characterization, and antioxidant activities of three Cu(II) complexes with Schiff-base ligands

Cu(II) complexes of three bis(pyrrol-2-yl-methyleneamine) ligands were synthesized and characterized by elemental analyses, mass spectra, and IR spectra. X-ray diffraction analysis shows that [CuL³]₂ is a dinuclear complex with an extremely distorted square-planar geometry. Furthermore, the antioxidant activities of the compounds have been investigated. The electrochemical properties of the Cu(II) complexes have also been studied by cyclic voltammetry. The Cu(II) complexes show similar superoxide dismutase (SOD) activity compared with that of the native Cu, Zn-SOD.     

A novel polymeric ionic liquid-coated magnetic multiwalled carbon nanotubes for the solid-phase extraction of Cu,Zn-superoxide dismutase

A novel magnetic adsorbent, benzyl groups functionalized imidazolium-based polymeric ionic liquid (PIL)-coated magnetic multiwalled carbon nanotubes (MWCNTs) (m-MWCNTs@PIL), has been successfully synthesized and applied for the extraction of Cu, Zn-superoxide dismutase (Cu, Zn-SOD). The m-MWCNTs@PIL were characterized by X-ray diffraction (XRD), Fourier transform infrared spectrometry (FT-IR), thermal gravimetric analysis (TGA), field emission scanning electron microscopy (FESEM), vibrating sample magnetometer (VSM) and zeta-potential nanoparticles. In this method, the m-MWCNTs@PIL could interact with Cu, Zn-SOD through hydrogen bonding, π-π and electrostatic interactions. The extraction performance of the m-MWCNTs@PIL in the magnetic solid-phase extraction (MSPE) procedure was investigated, coupled with the determination by UV–vis spectrophotometer. Compared with m-MWCNTs@IL and m-MWCNTs, the m-MWCNTs@PIL exhibited the highest extraction capacity of 29.1 mg/g for Cu, Zn-SOD. The adsorbed Cu, Zn-SOD remained high specific activity after being eluted from m-MWCNTs@PIL by 1 moL/L NaCl solution. Besides, the m-MWCNTs@PIL could be easily recycled and successfully employed in the extraction of Cu, Zn-SOD from real samples. Under the optimal conditions, the precision, repeatability and stability of the proposed method were investigated and the RSDs were 0.29%, 1.68% and 0.54%, respectively. Recoveries were in the range of 82.7–102.3%, with the RSD between 3.47% and 5.35%. On the basis of these results, the developed method has great potential in the extraction of Cu, Zn-SOD or other analytes from biological samples.     

Isoelectric buffers, part 3: determination of pKa and pI values of diamino sulfate carrier ampholytes by indirect UV-detection capillary electrophoresis

Ampholytes with close pK(a) values (i.e., good carrier ampholytes (CAs)) are needed as buffers in pH-biased isoelectric trapping (IET) separations. The syntheses of two families of such good CAs were reported recently. Members of the family of diamino sulfate ampholytes (first series) had pI values in the 5.7 < pI < 9.0 range. Members of the family of quaternary ammonium dicarboxylic acid ampholytes (second series) had pI values in the pI < 4.3 range. To further characterize the diamino sulfate ampholytes, their effective mobilities were measured by indirect UV-absorbance detection capillary electrophoresis in a series of background electrolytes (BGEs) with different pH values. The pK(a) and limiting ionic mobility values of the CAs were obtained by fitting these mobility values, as a function of the pH and the ionic strength of the BGEs, to the theoretical mobility expression. These diamino sulfates complete the list of CAs suitable for IET separations.