Determination of metal-cofactors in respiratory chain complexes by total-reflection X-ray fluorescence spectrometry (TXRF)

Total Reflection X-Ray Fluorescence Spectrometry (TXRF) offers many advantages for the detection of trace elements in enzymes as compared to other well known analytical techniques like flame-AAS or ICP-AES because of the significantly smaller amounts of sample required. Without any decomposition, elements like Fe, Ni, Cu, Zn, Mn and Mo could be determined with high accuracy, in spite of the large bio-organic matrix. Besides the metals also sulfur can be determined in protein samples. The two terminal oxidases, cytochrome c oxidase and quinol oxidase, isolated from the soil bacterium Paracoccus denitrificans, were transferred from their usual salt buffer into a solution of 100 mmol/L tris(hydroxymethyl)aminomethane (TRIS) acetate containing an appropriate detergent. By this procedure an improved signal/noise ratio is attained. The data for cytochrome c oxidase are in good agreement with values obtained by ICP-AES. Further results of quinol oxidase, which has different element ratios, also fit the expected values. The investigations lead to the conclusion that the method is well suited for the quantitative determination of metals in enzymes, and in particular their molar ratios, and requires only small amounts of the biological sample without any extensive pretreatment.     

Determination of metal-cofactors in enzyme complexes by total-reflection X-ray fluorescence spectrometry

The determination of metal-cofactors and their molar concentrations is an important requirement for the characterisation of metalloproteins and a challenge regarding the capabilities of trace analytical methods. In this respect, total-reflection X-ray fluorescence spectrometry offers many advantages for the determination of trace elements in enzymes, as compared to other well known analytical techniques such as flame atomic absorption spectrometry or inductively coupled plasma atomic emission spectrometry (ICP-AES), because of the significantly smaller amounts of sample required. Without any decomposition, elements like P, S, Fe, Ni, Cu, Zn, Mn and Mo could be determined with high accuracy, in spite of the large bio-organic matrix. The enzymes (polysulphide reductase and hydrogenase of the rumen bacterium Wolinella succinogenes, and the cytochrome c oxidase and quinol oxidase of the soil bacterium Paracoccus denitrificans) were transferred from their usual salt-buffer into a solution of 100 mmol l⁻¹ tris(hydroxymethyl)aminomethane (tris)-acetate containing an appropriate detergent. By this procedure, an improved signal-to-noise ratio is obtained. The polysulphide reductase was found to contain copper as a hitherto existing unknown cofactor. The enzyme contains a stretch of amino acids that are typical of copper proteins and thus confirm the presence of this element. Furthermore, the data concerning cytochrome c oxidase from Paracoccus denitrificans are in good agreement with published values obtained by ICP-AES. Also, results from measurements with the quinol oxidase from the same bacterium agree with the expected values. The investigations lead to the conclusion that the method is well suited to the quantitative determination of metals in enzymes, in particular their molar fractions, and requires only small amounts of the biological sample without any extensive pretreatment. © 1997 Elsevier Science B.V.     

Distribution and Transition of Native and Completely Unfolded Conformations in the Unfolding of Bovine Heart Cytochrome c Induced by Urea and Guanidine Hydrochloride

The unfolding of bovine heart cytochrome c induced by urea and guanidine hydrochloride was first studied through intrinsic fluorescence emission spectra and fluorescence phase diagram and the results showed that both of them separately followed a two‐state model. As the simplest sample of the unfolding of protein molecules induced by denaturants, an equation was presented to show the effect of the denaturant concentrations in denaturation solution on the residual activity ratios of bovine heart cytochrome c in their two‐state unfolding. There are two characteristic unfolding parameters K and m in this equation. The former is the thermodynamic equilibrium constant of the unfolding of bovine heart cytochrome c induced by denaturants, the latter is the number of denaturant molecules associated with a bovine heart cytochrome c molecule during the unfolding procedure, and through them the distribution and transition of native and completely unfolded bovine heart cytochrome c conformations under different concentrations of urea or guanidine hydrochloride in denaturation solution can be accurately described.     

The indirect coulometric titration of cytochrome c oxidase with cytochrome c

The indirect coulometric titration of cytochrome c oxidase and dioxygen using cytochrome c as a mediator is described. Results of both the indirect coulometric titrations and the cyclic voltammetric experiments reported herein verify that the reaction mechanism involves the catalytic regeneration of the electroactive species, the cytochrome c mediator, with the selective reduction of cytochrome c oxidase alone. During the indirect coulometric titrations dioxygen is reduced to water only by cytochrome c oxidase and not by either direct reduction at the electrode surface or reaction with cytochrome c. This system utilizes the electron transfer selectivity of cytochrome c for cytochrome c oxidase over dioxygen and offers a means by which the reaction of cytochrome c oxidase and dioxygen can be examined.     

Sequential determination of tin, arsenic, bismuth and antimony in marine sediment material by inductively coupled plasma atomic emission spectrometry using a small concentric hydride generator and L-cysteine as prereductant

A hydride generation system using a small concentric hydride generator combined with inductively coupled plasma atomic emission spectrometry (ICP-AES) was established to determine tin, arsenic, bismuth and antimony in a marine sediment material with L-cysteine as a pre-reductant. Influences of concentrations of three kinds of acids (HCl, HNO₃ and HClO₄), L-cysteine, and sodium tetrahydroborate(III) as well as sodium hydroxide were investigated. The interferences from transition ions were found to be insignificant for determination of the four elements in presence of L-cysteine. Under optimized conditions the detection limits were 0.6 ng/mL for arsenic(III), 0.8 ng/mL for antimony(III), 1.7 ng/mL for tin(IV), and 1.2 ng/mL for bismuth(III). The method was applied to determine the four elements in standard marine sediment materials and the results were in agreement with certified values. Received: 4 September 1997 / Revised: 14 October 1997 / Accepted: 7 November 1997     

Light-induced absorbance changes in cell-free extracts of Neurospora crassa.

Abstract— The mycelium of Neurospora crassa was ground and extracted with buffer and separated into a soluble supernatant fraction and a particulate fraction by centrifugation. Both fractions were examined for light-induced absorbance changes. Irradiation of the supernatant fraction caused a reversible photooxidation of cytochrome c which was inhibited by sodium azide or by dialysis. The action spectrum for the photooxidation showed that the response was mediated by an endogenous flavin. The photooxidation of cytochrome c, lost by dialysis, could be restored by adding flavin mononucleotide. Irradiation of the particulate fraction with blue light caused a reversible photoreduction of cytochrome c and cytochrome oxidase and, in some samples, of cytochrome b as well. The supernatant fraction showed photooxidation of cytochrome c rather than the more usual photoreductive changes because of the presence of super-oxide dismutase activity.     

Determination of trace amounts of Galaxolide? (HHCB) by HPLC

An analytical method for the determination of trace amounts of Galaxolide^? (HHCB) by HPLC is presented. It is based on the separation of HHCB in different samples by a C18-column and a gradient elution by water and acetonitrile, both containing some acetic acid. The detection is carried out with a combination of a fast scanning UV detector and a fast scanning fluorescence detector. The fluorescence detection limit is 5 μg/L HHCB without sample pretreatment. HHCB adsorbed on solids or suspended solids can be extracted by ethanol. Received: 17 October 1997 / Revised: 24 November 1997 / Accepted: 24 November 1997     

CeVO4 Nanozymes Catalyze the Reduction of Dioxygen to Water without Releasing Partially Reduced Oxygen Species

In this study, we report a remarkably active CeVO₄ nanozyme that functionally mimics cytochrome c oxidase (CcO), the terminal enzyme in the respiratory electron transport chain, by catalyzing a four‐electron reduction of dioxygen to water. The nanozyme catalyzes the reaction by using cytochrome c (Cyt c), the biological electron donor for CcO, at physiologically relevant pH. The CcO activity of the CeVO₄ nanozymes depends on the relative ratio of surface Ce³⁺/Ce⁴⁺ ions, the presence of V⁵⁺ and the surface‐Cyt c interactions. The complete reduction of oxygen to water takes place without release of any partially reduced oxygen species (PROS) such as superoxide, peroxide and hydroxyl radicals.     

The dual role of cytochrome c2 in the facultative phototrophic bacteriumRhodopseudomonas capsulata

Succinate oxidation is stimulated by addition of cytochrome c₂ in cytochrome c₂-deficient spheroplasts from the M6-mutant and from the wild type strain ofRhodopseudomonas capsulata. Inhibition of the alternative oxidase in the wild type by CO facilitates this observation. The finding confirms a dual role of cytochrome c₂, in photosynthetic and in respiratory electron transport.

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Die doppelte Rolle von Cytochrome c₂ in Rhodopseudomonas capsulata

Zusammenfassung Die Oxidation von Succinat wird durch Zugabe von Cytochrom c₂ zu Cytochrom c₂-defizienten Sphäroplasten der Mutante M6 und des Wildtyps vonRhodopseudomonas capsulata stimuliert. Die Hemmung der alternativen Oxidase durch CO im Wildtyp erleichtert diese Beobachtung. Der Befund bestätigt die Doppelrolle von Cytochrom c₂ im photosynthetischen und respiratorischen Elektronentransport.

     

Flavin-mediated photoreactions in artificial systems: a possible model for the blue-light photoreceptor pigment in living systems.

Abstract— The photoreduction of cytochrome c and the photostimulation of oxygen uptake were studied in solutions of flavin and cytochrome as a possible model system for similar photoreactions which have been observed in vivo. Light causes the photoreduction of the flavin. Under aerobic conditions the photoreduced flavin reacts with oxygen to form the superoxide anion which in turn can reduce cytochrome c. Dismutation of the superoxide anions forms hydrogen peroxide which mediates the dark oxidation of the photoreduced cytochrome. Superoxide formation and dismutation also account for the light-induced oxygen uptake. Action spectra confirm the role of flavin in the photoreduction of cytochrome c and the photostimulation of oxygen uptake. Under anaerobic conditions the photoreduced flavin reduces cytochrome c directly. In the presence of an electron donor only catalytic amounts of flavin are required. In the absence of an added electron donor flavin itself can act as the electron donor if substrate amounts are present. Azide inhibits all of these flavin-mediated photoresponses. Azide also inhibits the photoreduction of cytochrome b which occurs in the mycelium of Newospora.     

Determination of Cd, Cu, Pb and Sb in environmental samples by ICP-AES using polyaniline for separation

The anion exchange properties of polyaniline for Cd, Cu, Pb and Sb in potassium iodide were studied. The analytes converted into anionic complexes by KI (0.03–0.96 mol/L) in HCl were adsorbed on polyaniline and eluted with HNO₃. The optimum conditions for adsorption and elution were determined. Quantitative recoveries were obtained for Cd, Cu and Pb, whereas, the recoveries for Sb were about 75%. This separation procedure was used with subsequent ICP-AES determination for Cd, Cu, and Pb in NIST-coal fly ash (1633b) and a sea plant with an R.S.D of 5% (n = 5). Received: 13 November 1997 / Revised: 2 February 1998 / Accepted: 7 February 1998     

Study and application of a method for the determination of metallic elements by ICP-AES with preconcentration on an active carbon-silica gel microcolumn in a FI system

Using active carbon-silica gel as adsorbent and sodium diethyldithiocarbamate (NaDDTC) as chelating reagent in a flow injection system, an ICP-AES method has been developed for preconcentration and determination of trace metallic elements in high purity rare earth oxides. The experimental parameters such as pH, flow rate, amount of adsorbent, length of reaction coil and eluent acidity were optimized. At pH 4.6 Al,Cr,Cu,Fe,Pb,V,Zn could be preconcentrated, and subsequently determined by ICP-AES. This method can eliminate matrix effects. Its enrichment factors were 8.1–12.6 with detection limits in the ng/mL range and RSD of 2.3–5.0. This method was applied to the analysis of high purity of La₂O₃ and Eu₂O₃ with satisfactory results. Received: 24 April 1997 / Revised: 22 September 1997 / Accepted: 7 October 1997     

Study and application of a method for the determination of metallic elements by ICP-AES with preconcentration on an active carbon-silica gel microcolumn in a FI system

Using active carbon-silica gel as adsorbent and sodium diethyldithiocarbamate (NaDDTC) as chelating reagent in a flow injection system, an ICP-AES method has been developed for preconcentration and determination of trace metallic elements in high purity rare earth oxides. The experimental parameters such as pH, flow rate, amount of adsorbent, length of reaction coil and eluent acidity were optimized. At pH 4.6 Al,Cr,Cu,Fe,Pb,V,Zn could be preconcentrated, and subsequently determined by ICP-AES. This method can eliminate matrix effects. Its enrichment factors were 8.1–12.6 with detection limits in the ng/mL range and RSD of 2.3–5.0. This method was applied to the analysis of high purity of La₂O₃ and Eu₂O₃ with satisfactory results. Received: 24 April 1997 / Revised: 22 September 1997 / Accepted: 7 October 1997     

Nutritional and Environmental Properties of Algal Products Used in Healthy Diet by INAA and ICP-AES

Spirulina platensis alga sampled in the Caribbean Sea and seven other commercial algal products available on the Italian market of different origin and aspect, have been analyzed by instrumental neutron activation analysis (INAA) and inductively coupled plasma-atomic emission spectrometry (ICP-AES). By neutron irradiation and -ray spectrometry (INAA), as many as 20 elements could be measured instrumentally without any chemical treatment. Cu, Mg, Mn and Pb were determined after dissolution of the sample by ICP-AES. The cross-checking of the data, specifically by comparing those of Ca, Cr, Fe and Zn, obtained by the two techniques was found to be in good agreement. Special attention from analytical and nutritional point of view has been devoted to the toxic metals. The measurements have been carried out employing the reference algal material prepared by International Atomic Energy Agency (IAEA).     

Determination of the cadmium and copper content inherent to metallothionein

The reliability of the voltammetric determination of the cadmium and copper content (at pH 1.0), inherent to metallothionein (MT) isolated from the digestive gland of Mytilus galloprovincialis, was investigated. An artifact signal enhancement of copper, caused by the cupric-thionein complex adsorption at the mercury electrode, was established. This artifact was removed by UV-digestion of the sample for 15–20 h prior to analysis. A similar artifact was not detected for cadmium, because at this pH the cadmium-thionein complex has dissociated, and cadmium exists in the ionic form. Therefore, the voltammetric analysis of the cadmium content can be performed directly at pH 1.0, without prior UV-digestion of the sample. Received: 4 August 1997 / Revised: 12 November 1997 / Accepted: 16 November 1997     

Electrochemical Determination of Superoxide Based on Cytochrome c Immobilized on DDAB‐Modified Powder Microelectrode

Abstract

Cytochrome c was immobilized at didodecyldimethylammonium bromide (DDAB)‐modified powder microelectrode and presented quasi‐reversible electrochemistry. The apparent surface coverage of cytochrome c is greatly enhanced by using powder microelectrode technique, which is 1.21×10^?8 mol/cm², more than one to three orders of magnitude larger than that obtained with thiol and DNA‐modified Au electrode. The cytochrome c modified powder microelectrode was applied for the amperometric determination of superoxide generated by the reaction of hypoxanthine with xanthine oxidase (XOD) in the presence of dioxygen.

The detection sensitivity of the modified powder microelectrode is 0.74 µA/cm² µM, which is larger than that reported in previous publications. The detection limit of the modified powder microelectrode (PME) is 0.5 µM, and the linear detection range is 0.86～5.93 µM (values of the concentration are all in terms of hypoxanthine concentration in the solution).     

Electrocatalysis of the hydrogen production by [Fe] hydrogenase from Desulfovibrio vulgaris Hildenborough

Hydrogenases are enzymes which catalyze hydrogen production/consumption reactions. In this paper, the [Fe] hydrogenase from Desulfovibrio vulgaris Hildenborough is shown to catalyze the direct hydrogen production from protons, in the absence of any promoter, at a basal pyrolytic graphite electrode using cyclic voltammetry techniques. The effect of several parameters upon catalytic current is investigated: pH, hydrogenase concentration, ionic strength, competition with another protein (bovine serum albumin), cycling repetition, mode of electrode polishing. The extent and efficiency of the hydrogenase electroactivity are examined in the presence of either an artificial electron carrier (methylviologen) or a physiological partner cytochrome c₃ isolated from the same bacterial strain. Results suggest that the electron-transfer process is essentially controlled by a complex between both hydrogenase and cytochrome c₃. Electrocatalysis appears to be largely governed by the adsorption of hydrogenase on the electrode surface very likely involving hydrophobic interactions.     

Determination of trace elements in olive oil by ICP-AES and ETA-AAS: A pilot study on the geographical characterization

The determination of trace elements in edible oils is important because of both the metabolic role of metals and possibilities for adulteration detection and oil characterization.The most commonly used techniques for the determination of metals in oil samples are inductively coupled plasma atomic emission spectrometry (ICP-AES) and atomic absorption spectrometry (AAS). For this study, a microwave assisted decomposition of the olive oil in closed vessels using a mixture of nitric acid and hydrogen peroxide was applied as sample preparation.The low achievable LODs enable the determination by ICP-AES of even very low concentrations of most elements of interest. The proposed ICP-AES method permits the determination of Ca, Fe, Mg, Na, and Zn in olive oils. Elements present in small amounts (Al, Co, Cu, K, Mn, Ni) were measured by ETA-AAS in the same sample digest. The concentrations of Al, Co, Cu, K, Mn, and Ni were in the range from 0.15 to 1.5 μg/g and differ according to the geographical origin of the oils. For the amounts of Fe, Mg, Na, and Zn in the samples, no significant differences according to the geographical origin of the oils could be observed, the mean concentrations being 15.31, 3.26, 33.10, and 3.39 μg/g, respectively. The Ca content varies in the range of 1.3 to 9.0 μg/g.The dependency of the trace elemental content of olive oils on their geographical origin can be used for their local characterization.     

Direct and Cytochrome c Mediated Electrochemistry of Bilirubin Oxidase on Gold

Direct electron transfer (DET) of bilirubin oxidase from Myrothecium verrucaria (BOD) was established on promoter‐modified gold electrodes. The electrochemical behavior of the enzyme in solution was studied by means of cyclic voltammetry evaluating the biocatalytic reduction of dioxygen. The reaction of BOD at Au electrodes was shown to be efficient only at low pH. In addition, a novel interaction between BOD and cytochrome c (cyt.c) was found. It was shown that BOD efficiently accepts cyt.c as an electron donor in both cases when cyt.c is in solution and electrostatically adsorbed. The results suggest that cyt.c can play the role of a mediator facilitating electron transfer in a pH range where no DET could be observed between the enzyme and the electrode. For the interaction between cyt.c and BOD in solution the reaction kinetics has been studied electrochemically and spectrophotometrically.     

Direct analysis of solid samples by GFAAS – determination of trace heavy metals in barytes

Solid sampling graphite furnace atomic absorption spectroscopy (SS-GFAAS) has been used for the determination of traces of heavy metals (Cd, Pb, Cu, Cr, Ni, V and As) in barytes over a wide concentration range, e.g. Cd from 0.023 to 27.0 μg/g and Pb from 1.54 to 3509 μg/g.The necessity of determining heavy metals in commercial barytes (naturally occurring barium sulfate), a mineral important to the oil industry because of its use in drilling muds, is discussed. The problems presented by the analysis of this difficult matrix are elegantly solved by using SS-GFAAS for the direct determination of heavy metals. A high-performance graphite furnace AAS with D₂-background correction system and a transversely heated graphite atomizer was used for the investigations. The spectrometer was combined with a mechanical sampling module and an ultramicrobalance. The transfer of solid samples (sample weights 0.031–0.686 mg) into the atomizer was carried out by using an optimized graphite platform as the sample carrier. Calibration curve techniques and standard addition methods were employed using external standards (CRMs). Problems associated with signal deformations like multiple peaks, tailing or shoulders are also discussed and possibilities to solve the problems are given. The influence of the homogeneity of solid samples on the precision and accuracy are shown in a real example. The results obtained by SS-GFAAS were compared with results by other methods like X-ray fluorescence spectroscopy (XRF) and flame AAS after aqua regia microwave extraction. This study has demonstrated that SS-GFAAS is a very powerful and easy-to-use method for quick and accurate analysis of barytes. Received: 9 November 1998 / Revised: 29 January 1999 / Accepted: 2 February 1999