Metal ions-dependent peroxidase and oxidoreductase activities of polyclonal IgGs from the sera of Wistar rats

We present evidence showing that a small fraction of electrophoretically homogeneous IgGs from the sera of healthy Wistar rats is bound with several different Me2+ ions and oxidizes 3,3'-diaminobenzidine through a peroxidase activity in the presence of H2O2 and through an oxidoreductase activity in the absence of H2O2. During purification on Protein A-Sepharose and gel filtration, the polyclonal IgGs partially lose the Me2+ ions. Therefore, in the absence of external metal ions, the specific peroxidase activity of IgGs from the sera of different rats varied in the range 1.6-26% and increased up to 13-198% after addition of Fe2+ or Cu2+ ions as compared with horseradish peroxidase (HRP, taken for 100%). The oxidoreductase activity of HRP is 24-fold lower than its peroxidase activity, while oxidoreductase and peroxidase activities of IgGs are comparable. Oxidoreductase activities of different IgGs in the absence of external metal ions varied from 22 to 800%, and in the presence of Fe2+ or Cu2+ ions, from 37 to 1100% in comparison with the HRP oxidoreductase activity (100%). Chromatography of the IgGs on Chelex-100 leads to the adsorption of a small IgG fraction bound with metal ions and to its separation to many different subfractions demonstrating various affinities to the chelating resin and increased levels of the specific oxidoreductase and peroxidase activities. Antioxidant enzymes such as superoxide dismutases, catalases, and glutathione peroxidases are known to represent critical defense mechanisms for preventing oxidative modifications of DNA, proteins, and lipids. Peroxidase and oxidoreductase activity of antibodies may play an important role in the protection of organisms from oxidative stress and toxic compounds.     

Diversity of DNA‐hydrolyzing antibodies from the sera of autoimmune‐prone MRL/MpJ‐lpr mice

It has been shown for the first time that polyclonal IgG abzymes (Abzs) with DNase activity from the sera of autoimmune‐prone MRL/MpJ‐lpr mice can be separated by isoelectric focusing into many subfractions having the isoelectric points (pI) from 4.5 to 9, with the maximal activity for Abzs with pI = 6.5–9.0. Affinity chromatography on DNA‐cellulose separated DNase IgGs into many subfractions demonstrating a range of affinities for DNA and different levels of the relative DNase activities (RDA) due to intrinsically bound metals and after addition of external Mg²⁺, Mn²⁺, Ca²⁺, and Mg²⁺+Ca²⁺. Some fractions significantly increase RDAs in the presence of external ions (Mg²⁺+Ca²⁺ > Mg²⁺ > Mn²⁺ > Ca²⁺), while each of this cofactor can also inhibit or have no influence on the RDAs of another fractions. It is known that complexes of DNA with histones and other proteins of apoptotic cells are the primary immunogens in systemic lupus erythematosus (SLE). Bovine serum albumin (BSA) and methylated BSA (mBSA) increase the RDAs of only some fractions, while have no effect or inhibit other IgG fractions. The ratio of the RDAs in the presence of all metal ions, BSA, and mBSA was individual for every abzyme fraction. Mn²⁺ and Ca²⁺ stimulated accumulation of only relaxed form of supercoiled DNA (scDNA) in the case of all subfractions, while in the presence of Mg²⁺ antibodies (Abs) of some subfractions (and in the presence of Mn²⁺ +Ca²⁺ all subfractions) produced relaxed DNA (rDNA) and linear DNA (linDNA) in a variable extent. The data obtained show that the polyclonal Abzs of mice may be a cocktail of Abs directly to DNA, RNA, and their complexes with proteins and anti‐idiotypic Abs to active centers of different nucleases. The diversity of the physicochemical and kinetic characteristics of the Abzs seems to be significantly widened when pre‐diseased mice spontaneously develop the disease. Copyright © 2010 John Wiley & Sons, Ltd.     

Substrate specificity of IgGs with peroxidase and oxidoreductase activities from sera of patients with systemic lupus erythematosus and multiple sclerosis

The analysis of IgGs to protect humans from oxidative stress through oxidation of harmful compounds was carried out. We have compared here for the first time peroxidase (in the presence of H₂O₂) and oxidoreductase (in the absence of H₂O₂) activities of IgGs from sera of healthy humans and patients with systemic lupus erythematosus (SLE) and multiple sclerosis (MS). In addition, substrate specificity of SLE and MS IgG preparations in the oxidation of different compounds was analyzed: 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS), 3,3′‐diaminobenzidine (DAB), homovanillic acid (HVA), o‐phenylenediamine (OPD), α‐naphthol, 3‐amino‐9‐ethylcarbazole (AEC), p‐hydroquinone (pHQ), and adrenaline. IgGs of healthy humans and SLE and MS patients oxidized DAB, ABTS, and OPD due to their peroxidase and oxidoreductase activities, while other compounds were substrates of IgGs only in the presence of H₂O₂: adrenaline was not oxidized by both activities of IgGs. The average SLE IgGs peroxidase activity increased statistically significant in comparison with abzymes from healthy humans in the order (‐fold): OPD (1.2) <  DAB (1.7) < α‐naphtol (2.2) ≤ AEC (2.4) < ABTS (4.5) < 5‐ASA (10.6), while with oxidoreductase activity: OPD (1.8) ≤ DAB (2.1‐fold) < ABTS (5.0). Only HVA was oxidized by IgGs with peroxidase activity of healthy donors faster than by SLE (1.3‐fold) and MS abzymes (2.4‐fold). In the oxidation of several substrates, only three IgGs of MS patients were used. The data speak of a tendency to increase the peroxidase and oxidoreductase activities of MS IgGs in comparison with healthy donors, but to a lesser extent: OPD (1.1 to 1.2‐fold) ≤ ABTS (1.2 to 1.8‐fold). It was shown that development of SLE and MS leads to increase in peroxidase and oxidoreductase activities of IgGs toward most of classical substrates. Thus, abzymes can serve as an additional factor of reactive oxygen species detoxification protecting of patients with SLE and MS from some harmful compounds somewhat better than healthy peoples.     

Effect of CoCl2 on the content of different metals and a relative activity of DNA‐hydrolyzing abzymes in the blood plasma of mice

Cobalt is a transition metal and an essential trace element that is required for vitamin B₁₂ biosynthesis, enzyme activation, and so on but is toxic in high concentrations. It was shown that the content of different elements in the plasma of 2‐month‐old BALB/c mice (control group) decreased in the following order: Ca > Mg > Si > Fe > Zn > Cu ≥ Al ≥ B. The treatment of mice with CoCl₂ did not appreciably change the relative content of Ca, Cu, and Zn, but a significant increase in the content of B (2.3‐fold), Mg (1.5‐fold), Al and Fe (2.1‐fold), and Si (3.4‐fold) was found. The treatment of mice led to a 2.2‐fold decrease in the concentration of the total blood protein and a 1.7 ± 0.2‐fold decrease of total immunoglobulin Gs (IgGs). Deoxyribonuclease IgGs corresponding to mice treated (t‐IgGs) and non‐treated (nt‐IgGs) with CoCl₂ contained intrinsically bound metal ions; these IgGs hydrolyzed DNA with very low activity but were not active in the presence of ethylenediaminetetraacetic acid or after Ab dialysis against ethylenediaminetetraacetic acid. The average RAs of deoxyribonuclease nt‐IgGs increased after addition of external metal ions in the following order: Zn²⁺ < Ca²⁺ < Cu²⁺ < Fe²⁺ < Mn²⁺ < Mg²⁺ < Co²⁺ < Ni²⁺. Interestingly, t‐IgGs demonstrated lower activities than those for nt‐IgGs either in the absence of external metal ions (2.7‐fold) or in the presence of Cu²⁺ (9.5‐fold) > Co²⁺ (5.6‐fold) > Zn²⁺ (5.1‐fold) > Mg²⁺ (4.1‐fold) > Ca²⁺ (3.0‐fold) > Fe²⁺ (1.3‐fold). However, the RAs of t‐IgGs were remarkably more active than nt‐IgGs in the presence of best activators of t‐IgGs Ni²⁺ (1.4‐fold) and especially Mn²⁺ (2.2‐fold). The data may be useful for an understanding of Co toxicity, its effect on the concentration of other metal ions, and a change of metal‐dependent specificity of Abzs. Copyright © 2012 John Wiley & Sons, Ltd.     

Metal dependent hydrolysis of β‐casein by sIgA antibodies from human milk

We present the first evidence that electrophoretically and immunologically homogeneous sIgAs purified from milk of healthy human mothers by chromatography on Protein A‐Sepharose and FPLC gel filtration contain intrinsically bound metal ions (Ca > Mg ≥ Al > Fe ≈ Zn ≥ Ni ≥ Cu ≥ Mn), the removal of which by a dialysis against ethylenediamine tetraacetic acid (EDTA) leads to a significant decrease in the β‐casein‐hydrolyzing activity of these antibodies (Abs). An affinity chromatography of total sIgAs on benzamidine‐Sepharose interacting with canonical serine proteases separates a small metalloprotease sIgA fraction (6.8 ± 2.4%) from the main part of these Abs with a serine protease‐like β‐casein‐hydrolyzing activity. The relative activity of this metalloprotease sIgA fraction containing intrinsically bound metal ions increases ～1.2–1.9‐fold after addition of external metal ions (Mg²⁺ > Fe²⁺ > Cu²⁺ ≥ Ca²⁺ ≥ Mn²⁺) but decreases by 85 ± 7% after the removal of the intrinsically bound metals. The metalloprotease sIgA fraction free of intrinsic metal ions demonstrates a high β‐casein‐hydrolyzing activity in the presence of individual external metal ions (Fe²⁺ > Ca²⁺ > Co²⁺ ≥ Ni²⁺) and especially several combinations of metals: Co²⁺ + Ca²⁺ < Mg²⁺ + Ca²⁺ < Ca²⁺ + Zn²⁺ < Fe²⁺ + Zn²⁺ < Fe²⁺ + Co²⁺ < Fe²⁺ + Ca²⁺. The patterns of hydrolysis of a 22‐mer oligopeptide corresponding to one of sIgA‐dependent specific cleavage sites in β‐casein depend significantly on the metal used. Metal‐dependent sIgAs demonstrate an extreme diversity in their affinity for casein‐Sepharose and chelating Sepharose, and interact with Sepharoses bearing immobilized monoclonal mouse IgGs against λ‐ and κ‐type light chains of human Abs. Possible ways of the production of metalloprotease abzymes (Abz) by human immune system are discussed. Copyright © 2010 John Wiley & Sons, Ltd.     

Oxidoreductase activities of polyclonal IgGs from the sera of Wistar rats are better activated by combinations of different metal ions

It was shown that IgGs purified from the sera of healthy Wistar rats contain several different bound Me2+ ions and oxidize 3,3'-diaminobenzidine through a H2O2-dependent peroxidase and H2O2-independent oxidoreductase activity. IgGs have lost these activities after removing the internal metal ions by dialysis against EDTA. External Cu2+ or Fe2+ activated significantly both activities of non-dialysed IgGs containing different internal metals (Fe > or = Pb > or = Zn > or = Cu > or = Al > or = Ca > or = Ni > or = Mn > Co > or = Mg) showing pronounced biphasic dependencies corresponding to approximately 0.1-2 and approximately 2-5 mM of Me2+, while the curves for Mn2+ were nearly linear. Cu2+ alone significantly stimulated both the peroxidase and oxidoreductase activities of dialysed IgGs only at high concentration (> or = 2 mM), while Mn2+ weakly activated peroxidase activity at concentration >3 mM but was active in the oxidoreductase oxidation at a low concentration (<1 mM). Fe2+-dependent peroxidase activity of dialysed IgGs was observed at 0.1-5 mM, but Fe2+ was completely inactive in the oxidoreductase reaction. Mg2+, Ca2+, Zn2+, Al2+ and especially Co2+ and Ni2+ were not able to activate dialysed IgGs, but slightly activated non-dialysed IgGs. The use of the combinations of Cu2+ + Mn2+, Cu2+ + Zn2+, Fe2+ + Mn2+, Fe2+ + Zn2+ led to a conversion of the biphasic curves to hyperbolic ones and in parallel to a significant increase in the activity as compared with Cu2+, Fe2+ or Mn2+ ions taken separately; the rates of the oxidation reactions, catalysed by non-dialysed and dialysed IgGs, became comparable. Mg2+, Co2+ and Ni2+ markedly activated the Cu2+-dependent oxidation reactions catalysed by dialysed IgGs, while Ca2+ inhibited these reactions. A possible role of the second metal in the oxidation reactions is discussed.     

Pyrophosphate as substrate for alkaline phosphatase activity: A convenient flow‐injection chemiluminescence assay

A sensitive and convenient flow‐injection chemiluminescence (FI‐CL) turn‐on assay for alkaline phosphatase (ALP) activity without any label and synthesis is developed. Cu²⁺ can catalyze the luminol–H₂O₂ CL reaction. Pyrophosphate (PPi) can chelate Cu²⁺ and therefore the Cu²⁺‐mediated luminol‐H₂O₂ CL reaction is inhibited. The addition of ALP can catalyze the hydrolysis of PPi into phosphate ions, Cu²⁺ is released and the chemiluminescence recovers. A detection limit of 1 mU/mL ALP is obtained.     

IgGs containing λ- and κ-type light chains and of all subclasses (IgG1-IgG4) from the sera of patients with autoimmune diseases and viral and bacterial infections hydrolyze DNA

We present the first evidence demonstrating that small fractions of IgGs of all four subclasses (IgG1–IgG4) from patients with viral (tick‐borne encephalitis), bacterial infections (streptococcal infection or erysipelas), and suppurative surgical infections caused by epidermal staphylococci as well as from patients with autoimmune diseases (systemic lupus erythematosus and multiple sclerosis) are catalytically active in the hydrolysis of supercoiled DNA. The hydrolysis of DNA was analyzed by agarose gel electrophoresis. The catalytic activities of nonfractionated IgGs increased in the following order: tick‐borne encephalitis < suppurative surgical infection < streptococcal infection < multiple sclerosis < systemic lupus erythematosus, whereas IgGs of healthy donors were inactive. However, the pools of antibodies corresponding to any particular disease were characterized by a specific ratio of IgGs of all four subclasses (IgG1–IgG4) and IgGs containing λ‐ and κ‐type light chains, and each of these subfractions of immunoglobulins demonstrated characteristic relative DNase activity. The relative activities of IgGs containing λ‐type light chains may on average be higher, lower, or comparable with those for IgGs with κ‐type light chains. The relative contributions of IgGs of different subclasses to the total activity of IgGs also varied widely in the case of various diseases: IgG1 (7%–45%), IgG2 (0.4%–73%), IgG3 (0%–12%), and IgG4 (9%–66%). Thus, immune systems of patients with different diseases can generate a variety of anti‐DNA abzymes of different types and with different catalytic properties, which can play an important role in the pathogenesis or protection from the development of these diseases. Copyright © 2012 John Wiley & Sons, Ltd.     

DNA-hydrolyzing activity of IgG antibodies from the sera of patients with tick-borne encephalitis

DNase autoantibodies (Abzs) can be found in the blood of patients with several autoimmune diseases, while the blood of healthy donors or patients with diseases with an insignificant disturbance of the immune status does not contain DNase Abzs. Here we present the first analysis of the DNase Abzs activity in the patients with tick-borne encephalitis (TBE). Several strict criteria have been applied to show that the DNase activity is an intrinsic property of IgGs from the sera of TBE patients but not from healthy donors. The relative activity of IgGs has been shown to vary extensively from patient to patient, but most of the preparations (91%) had detectable levels of the DNase activity. Polyclonal DNase IgGs were not active in the presence of EDTA or after a dialysis against EDTA, but could be activated by several externally added metal ions, with the level of activity decreasing in the order Mn²⁺ + Ca²⁺ ≥ Mn²⁺+ Mg²⁺ ≥ Mn²⁺ ≥ Mg²⁺ + Ca²⁺ ≥ Co²⁺ ≥ Mg²⁺ > Ca²⁺, while K⁺, Na⁺, Ni²⁺, Zn²⁺, and Cu²⁺ did not stimulate DNA hydrolysis. Affinity chromatography on DNA-cellulose separated the DNase IgGs into many subfractions with various affinities for DNA and very different levels of the relative activity. Possible reasons for catalytic diversity of polyclonal human Abzs are discussed.     

Diversity of integrase-hydrolyzing IgGs and IgMs from sera of HIV-infected patients

It was previously shown that small fractions of IgGs and IgMs from the sera of AIDS patients specifically hydrolyze only HIV integrase (IN) but not many other tested proteins. Here we present evidence showing that these IgGs and IgMs are extreme catalytically heterogeneous. Affinity chromatography on IN-Sepharose using elution of IgGs (or IgMs) with different concentration of NaCl and acidic buffer separated catalytic antibodies (ABs) into many AB subfractions demonstrating different values of K _m for IN and k _cat. Nonfractionated IgGs and IgMs possess serine-, thiol-, acidiclike, and metal-dependent proteolytic activity. Metal-dependent activity of abzymes increases in the presence of ions of different metals. In contrast to canonical proteases having one pH optimum, initial nonfractionated IgGs and IgMs demonstrate several optima at pH from 3 to 10. The data obtained show that IN-hydrolyzing polyclonal IgG and IgM of HIV-infected patients are cocktails of anti-IN ABs with different structure of the active centers possessing various affinity to IN, pH optima, and relative rates of the specific substrate hydrolysis.     

Multifunctional organic–inorganic composite luminescent nanospheres

A simple general strategy was successfully developed for the preparation of magnetic–luminescent multifunctional nanocomposites by incorporating fluorescent (pyrene) and magnetic (Fe₃O₄) components simultaneously into a poly(styrene‐co‐methacrylic acid) [poly(St‐co‐MAA)] copolymer matrix. The nanospheres so prepared were characterized using scanning electron microscopy (SEM), powder X‐ray diffraction (XRD) and Fourier transform infrared (FTIR) analysis. The prepared magnetic–fluorescent inorganic–organic nanocomposites have excellent magnetic and photoluminescent properties. They can be used in magnetic separation of trace amounts of sample, fluorescence detection and imaging applications, including magnetic resonance imaging (MRI) and fluorescence imaging. The fluorescence quenching of the nanospheres in the presence of different amounts of Cu²⁺ ions was also investigated. Under optimal experimental conditions, the relative fluorescence intensity of the composite nanosphere colloidal solution is proportional to the concentration of Cu²⁺ ions, which indicates that these multifunctional nanocomposites can be used for the magnetic separation and fluorescence detection of Cu²⁺ ions. Copyright © 2011 John Wiley & Sons, Ltd.     

A N‐(2‐hydroxyethyl)piperazine dangled 2,5‐diphenyl‐1,3,4‐oxadiazole‐based fluorescent sensor for selective relay recognition of Cu2+ and sulfide in water

A new 2,5‐diphenyl‐1,3,4‐oxadiazole‐based derivative (L) was synthesized and applied as a highly selective and sensitive fluorescent sensor for relay recognition of Cu²⁺ and S^2? in water (Tris–HCl 10 mM, pH = 7.0) solution. L exhibits an excellent selectivity to Cu²⁺ over other examined metal ions with a prominent fluorescence ‘turn‐off’ at 392 nm. L interacts with Cu²⁺ through a 1:2 binding stoichiometry with a detection limit of 4.8 × 10^–7 M. The on‐site formed L–2Cu²⁺ complex exhibits excellent selectivity to S^2? with a fluorescence ‘off–on’ response via a Cu²⁺ displacement approach. Copyright © 2016 John Wiley & Sons, Ltd.     

Activation of DNA-hydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr mice by different metal ions

We have shown previously that electrophoretically and immunologically homogeneous polyclonal IgGs from the sera of autoimmune-prone MRL mice possess DNase activity. Here we have analyzed for the first time activation of DNase antibodies (Abs) by different metal ions. Polyclonal DNase IgGs were not active in the presence of EDTA or after Abs dialysis against EDTA, but could be activated by several externally added metal (Me(2+)) ions, with the level of activity decreasing in the order Mn(2+)> or =Mg(2+)>Ca(2+)> or =Cu(2+)>Co(2+)> or =Ni(2+)> or =Zn(2+), whereas Fe(2+) did not stimulate hydrolysis of supercoiled plasmid DNA (scDNA) by the Abs. The dependencies of the initial rate on the concentration of different Me(2+) ions were generally bell-shaped, demonstrating one to four maxima at different concentrations of Me(2+) ions in the 0.1-12 mM range, depending on the particular metal ion. In the presence of all Me(2+) ions, IgGs pre-dialyzed against EDTA produced only the relaxed form of scDNA and then sequence-independent hydrolysis of relaxed DNA followed. Addition of Cu(2+), Zn(2+), or Ca(2+) inhibited the Mg(2+)-dependent hydrolysis of scDNA, while Ni(2+), Co(2+), and Mn(2+) activated this reaction. The Mn(2+)-dependent hydrolysis of scDNA was activated by Ca(2+), Ni(2+), Co(2+), and Mg(2+) ions but was inhibited by Cu(2+) and Zn(2+). After addition of the second metal ion, only in the case of Mg(2+) and Ca(2+) or Mn(2+) ions an accumulation of linear DNA (single strand breaks closely spaced in the opposite strands of DNA) was observed. Affinity chromatography on DNA-cellulose separated DNase IgGs into many subfractions with various affinities to DNA and very different levels of the relative activity (0-100%) in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. In contrast to all human DNases having a single pH optimum, mouse DNase IgGs demonstrated several pronounced pH optima between 4.5 and 9.5 and these dependencies were different in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. These findings demonstrate a diversity of the ability of IgG to function at different pH and to be activated by different optimal metal cofactors. Possible reasons for the diversity of polyclonal mouse abzymes are discussed.     

Effect of linear alkylbenzene sulfonate on Cu2+ removal by Spirulina platensis strain (FACHB‐834)

The removal efficiency of Cu²⁺ by Spirulina platensis (strain FACHB‐834), in viable and heat‐inactivated forms, was investigated in the presence and absence of linear alkylbenzene sulfonate (LAS). When the initial Cu²⁺ concentration was in the range of 0.5–1.5 mg · L^?1, a slight increase in growth rate of FACHB‐834 was observed. In contrast, when Cu²⁺ or LAS concentrations were at or higher than 2.0 or 6.0 mg · L^?1, respectively, the growth of FACHB‐834 was inhibited and displayed yellowing and fragmentation of filaments. The presence of LAS improved Cu²⁺ removal by ~20%, and accelerated attainment of Cu²⁺ retention equilibrium. For the 2‐ mg · L^?1 Cu²⁺ treatments, retention equilibrium occurred within 2 d and showed maximum Cu²⁺ removal of 1.83 mg · L^?1. In the presence of LAS, the ratio of extracellular bound Cu²⁺ to intracellular Cu²⁺ taken up by the cells was lower (1.05–2.26) than corresponding ratios (2.46–7.85) in the absence of LAS. The percentages of extracellular bound Cu²⁺ to total Cu²⁺ removal (both bound and taken up by cells) in the presence of LAS ranged from 51.2% to 69.3%, which was lower than their corresponding percentages (71.1%–88.7%) in the absence of LAS. LAS promoted biologically active transport of the extracellular bound form of Cu²⁺ into the cell. In contrast, the addition of LAS did not increase the maximum removal efficiency of Cu²⁺ (61.4% ± 5.6%) by heat‐inactivated cells compared to that of living cells (59.6% ± 6.0%). These results provide a theoretical foundation for designing bioremediation strategies using FACHB‐834 for use in surface waters contaminated by both heavy metals and LAS.     

Paramelaconite‐Enriched Copper‐Based Material as an Efficient and Robust Catalyst for Electrochemical Carbon Dioxide Reduction

A copper‐oxide‐based catalyst enriched with paramelaconite (Cu₄O₃) is presented and investigated as an electrocatalyst for facilitating electroreduction of CO₂ to ethylene and other hydrocarbons. Cu₄O₃ is a member of the copper‐oxide family and possesses an intriguing mixed‐valance nature, incorporating an equal number of Cu⁺ and Cu²⁺ ions in its crystal structure. The material is synthesized using a solvothermal synthesis route and its structure is confirmed via powder X‐ray diffraction, transmission electron microscope based selected area electron diffraction, and X‐ray photoelectron spectroscopy. A flow reactor equipped with a gas diffusion electrode is utilized to test a copper‐based catalyst enriched with the Cu₄O₃ phase under CO₂ reduction conditions. The Cu₄O₃‐rich catalyst (PrC) shows a Faradaic efficiency for ethylene over 40% at 400 mA cm^?2. At ?0.64 versus reversible hydrogen electrode, the highest C₂₊/C₁ product ratio of 4.8 is achieved, with C₂₊ Faradaic efficiency over 61%. Additionally, the catalyst exhibits a stable performance for 24 h at a constant current density of 200 mA cm^?2.     

The Effects of Copper (II) Ions on <Emphasis Type="Italic">Enterococcus hirae</Emphasis> Cell Growth and the Proton-Translocating F<Subscript>o</Subscript>F<Subscript>1</Subscript> ATPase Activity

Enterococcus hirae grow well under anaerobic conditions at alkaline pH (pH 8.0) producing acids by glucose fermentation. Bacterial growth was shown to be accompanied by decrease of redox potential from positive values (~+35 mV) to negative ones (~−220 mV). An oxidizer copper (II) ions (Cu²⁺) affected bacterial growth in a concentration-dependent manner (within the range of 0.05 mM to 1 mM) increasing lag phase duration and decreasing specific growth rate. These effects were observed with the wild-type strain ATCC9790 and the atpD mutant strain MS116 (with absent β subunit of F₁ of the F_oF₁ ATPase) both. Also ATPase activity and proton–potassium ions exchange were assessed with and without N,N′-dicyclohexylcarbodiimide (DCCD), inhibitor of the F_oF₁ ATPase. In both cases (DCCD ±), even low Cu²⁺ concentrations had noticeable effect on ATPase activity, but with less visible concentration-dependent manner. Changes in the number of accessible SH-groups were observed with E. hirae ATCC9790 and MS116 membrane vesicles. In both strains Cu²⁺ markedly decreased the number of SH-groups in the presence of K⁺ ions. The addition of ATP increased the amount of accessible SH-groups in ATCC9790 and decreased this number in MS116; Cu²⁺ blocked ATP-installed increase in SH-groups number in ATCC9790. H⁺–K⁺-exchange of bacteria was markedly inhibited by Cu²⁺, but stronger effects were detected together with DCCD. Moreover, discrimination between Cu²⁺ and other bivalent cation—Ni²⁺ was shown. It is suggested that Cu²⁺ ions inhibit E. hirae cell growth by direct affect on the F_oF₁ ATPase leading to conformational changes in this protein complex and decrease in its activity.     

Some metals inhibit the glutathione S‐transferase from Van Lake fish gills

Glutathione S‐transferases (GSTs) are the superfamily of multifunctional detoxification isoenzymes and play important role cellular signaling. The present article focuses on the role of Cd²⁺, Cu²⁺, Zn²⁺, and Ag⁺ in vitro inhibition of GST. For this purpose, GST was purified from Van Lake fish (Chalcalburnus tarichii Pallas) gills with 110.664 EU mg^?1 specific activity and 79.6% yield using GSH‐agarose affinity chromatographic method. The metal ions were tested at various concentrations on in vitro GST activity. IC₅₀ values were found for Cd⁺², Cu⁺², Zn⁺², Ag⁺ as 450.32, 320.25, 1510.13, and 16.43 μM, respectively. K _i constants were calculated as 197.05 ± 105.23, 333.10 ± 152.76, 1670.21 ± 665.43, and 0.433 ± 0.251 μM, respectively. Ag⁺ showed better inhibitory effect compared with the other metal ions. The inhibition mechanisms of Cd²⁺ and Cu²⁺ were non‐competitive, whereas Zn²⁺ and Ag⁺ were competitive. Co²⁺, Cr²⁺, Pb²⁺, and Fe³⁺ had no inhibitory activity on GST.     

Affinity and catalytic heterogeneity of polyclonal myelin basic protein‐hydrolyzing IgGs from sera of patients with multiple sclerosis

Human myelin basic protein (hMBP)‐hydrolyzing activity was recently shown to be an intrinsic property of antibodies (Abs) from multiple sclerosis (MS) patients. Here, we present the first evidence demonstrating a significant diversity of different fractions of polyclonal IgGs (pIgGs) from MS patients in their affinity for hMBP and in the ability of pIgGs to hydrolyze hBMP at different optimal pHs (3–10.5). IgGs containing λ‐ and κ‐types of light chains demonstrated comparable relative activities in the hydrolysis of hMBP. IgGs of IgG1–IgG4 sub‐classes were analyzed for catalytic activity. IgGs of all four sub‐classes were catalytically active, with their contribution to the total activity of Abzs in the hydrolysis of hMBP and its 19‐mer oligopeptide increasing in the order: IgG1 (1.5–2.1%) < IgG2 (4.9–12.8%) < IgG3 (14.7–25.0%) < IgG4 (71–78%). Our findings suggest that the immune systems of individual MS patients generate a variety of anti‐hMBP abzymes with different catalytic properties, which can attack hMBP of myelin‐proteolipid shell of axons, playing an important role in MS pathogenesis.     

Studies on nucleic acids. VIII. Changes in the stability of DNA secondary structure by interaction with divalent metal ions

The melting temperature of a natural DNA is decreased in the presence of increasing amounts of copper ions, whereas other divalent metal ions stabilize the DNA secondary structure at low ionic strength. At 1.28 × 10^?4M, Cu²⁺ produces a decrease of T_m depending on base composition. At very low Cu²⁺ concentrations (0.5 Cu²⁺/2 DNA-P) a stabilization of the DNA conformation appears due to an interaction between Cu²⁺ and phosphate groups of the DNA molecule. In this case the normal trend of GC dependence of T_m exists similar to that with Na⁺ and Mg²⁺ as counterions. If copper ions are in excess, the observed destabilization is stronger for DNAs rich in guanine plus cytosine than for those rich in adenine plus thymine. A sharp decrease of T_m occurs between 0.5–0.8 Cu²⁺/2 DNA-P and 1.5 Cu²⁺/2 DNA-P. The breadth of the transition decreases at high Cu²⁺ concentration with further addition of copper ions. Denaturation and renaturation experiments indicate that Cu²⁺ ions exceeding the phosphate equivalents interact with the bases and reduce the forces of the DNA helix conformation. Evidence is presented, that the destabilization effect produced by Cu²⁺ is possibly due to an interaction with guanine sites of the DNA molecule.