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 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. Received: 17 June 1997 / Revised: 21 November 1997 / Accepted: 27 November 1997     

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.     

Thermal lens determination of cytochrome c and its NO complex

Spectrophotometric and thermal lens measurements showed that cw laser beam (450–530 nm, up to 100 mW) does not affect the absorption band of cytochrome c. Therefore, thermal lensing is used for determining cytochrome c (III) (c _min = 1 × 10^?7 mol/L at λ = 488.0 nm; c _min = 3 × 10^?8 mol/L at λ = 514.5 nm) and its active form, cytochrome c (II) (c _min = 1 × 10^?8 mol/L at λ = 514.5 nm). The enhancement of the sensitivity of determination of these species as compared with conventional spectrophotometry is more than two orders of magnitude. The optimal conditions for the formation of the NO complex of cytochrome c for its photometric determination were selected: the molar ratio of dodecyl sulfate (a modifying agent) to cytochrome c is 1: 30 at a working wavelength of 560 nm. When exposed to laser radiation, the nitrosyl complex of cytochrome c dissociates to form cytochrome c (III). The decomposition of this complex can be monitored by thermal lensing (514.5 nm) down to a level of 1 × 10^?7 mol/L.     

Revised structure of a catecholamide spermidine siderophore : From Paracoccus denitrificane

Evidence has been found for the presence of an oxazoline ring in a siderophore from Paracoccus denitrificans. This requires revision of the previously assigned structure for this compound.     

Electrochemical characterization of a superoxide biosensor based on the co-immobilization of cytochrome c and XOD on SAM-modified gold electrodes and application to garlic samples

This paper describes the characterization and optimization of an amperometric cytochrome c (cyt c)-based sensor for the determination of the antioxidant capacity of pure substances and natural samples. The cyt c and the xanthine oxidase (XOD) enzyme were co-immobilized on the electrode using the combination of several long-chain thiols. The self-assembled monolayer (SAM) was optimized in terms of composition and ratio between thiols. The immobilization protocol for both cyt c and XOD and the SAM formation time were evaluated through electrochemical methods, such as cyclic voltammetry (CV), square wave voltammetry (SWV), chronoamperometry (CA) and impedance spectroscopy (IS). Finally, the biosensor was applied to the determination of the antioxidant capacity of pure alliin and two compounds extracted from garlic bulbs.     

Production of L-phenylalanine from acetamidocinnamic acid usingBacillus sphaericus andParacoccus denitrificans

To establish an efficient production method for L-phenylalanine (L-Phe), the possibility of L-Phe production from acetamidocinnamic acid by a two-step enzyme reaction was investigated.Bacillus sphaericus N-7 (FERM-P no. 6746) was selected as an enzyme source for the acylase used to form phenylpyruvic acid from aceta midocinnamic acid.Paracoccus denitrificans IFO 12442 was selected as an aminotransferase source for the conversion of phenylpyruvic acid to L-Phe. By using both cell types, 0.46M L-Phe was produced in a molar yield of 92% from 0.5M acetamidocinnamic acid.     

Conformational transitions in the regulation of cytochrome c oxidase activity

Oxidation of cytochrome c, catalyzed by cytochrome oxidase embedded in artificial liposomes of high respiratory control ratio (between 5 and 9.5), has been studied by rapid mixing techniques, under which conditions the enzyme undergoes a limited number of turnovers (from 1 to 5). The time course of the reaction could be satisfactorily simulated by a procedure derived from the concerted two-state model of Monod-Wyman-Changeux.The bulk of data and the novel analytical approach confirm the proposal that cytochrome oxidase undergoes a transition from a fast-reacting to a slow-reacting form as a consequence of the electrochemical gradient built up across the phospholipidic bilayer, and substantiate the idea that the conformational change:

• •occurs as an all-or-none process after about one turnover irrespective of the molar ratio between substrate and enzyme, and
• •is not immediately correlated to the other well known transition from the resting to the pulsed form of the enzyme.

     

Direct determination of trace elements in niobium,tantalum and their oxides by inductively coupled plasma atomic emission spectrometry after microwave dissolution

Analytical schemes for the determination of trace elements in high-purity niobium, tantalum and their oxides are proposed. The schemes are based on microwave dissolution of the metals and oxides followed by inductively coupled plasma atomic emission spectrometry (ICP-AES) determination of impurities in the solutions. The possibilities of interelement and off-peak background corrections in ICP-AES analysis are discussed. The accuracy of the results obtained is confirmed by the determination of trace elements after a matrix sorption separation procedure. For a number of elements, a comparison of the results obtained by ICP-AES without and with the matrix separation procedure and by electrothermal atomic absorption spectrometry (ETAAS) shows good agreement. The limits of detection for direct ICP-AES determination are in the range 0.4*1.0 μg g⁻¹ for Ba, Ca, Fe, Mg, Mn, Y and La; between 2.0 and 10.0 μ g⁻¹ for B, Cd, Co, Cr, Cu, Hf, Mo, Na, Nb, Ni, Pb, Sr, Ti, Zr and Ta; and for K, Sb and W a detection limit of 20 μ g⁻¹ is achieved. The schemes proposed are intended for rapid routine analysis.     

Capillary zone electrophoresis with field enhanced sample stacking as a tool for targeted metabolome analysis of adenine nucleotides and coenzymes in Paracoccus denitrificans

The main aim of this work was to demonstrate the applicability of capillary zone electrophoresis in combination with field enhanced sample stacking in targeted metabolome analyses of adenine nucleotides – AMP, ADP, ATP, coenzymes NAD⁺, NADP⁺ and their reduced forms in Paracoccus denitrificans. Sodium carbonate/hydrogencarbonate buffer (100 mM, pH 9.6) with the addition of β‐CD at a concentration of 10 mM was found to be an effective BGE for their separation within 20 min. Besides this, special attention was paid to the development of the procedure for the extraction of specific metabolites from the bacterium P. denitrificans. This procedure was not only optimised to achieve the highest metabolite yields but also to obtain a sample that was fully compatible with the online preconcetration strategy used. The developed methodology was finally applied in a study of the bacterium P. denitrificans at various stages of the active respiratory chain.     

866 — Interaction of cationic drugs with lipids of the spinach thylakoid membrane

Adriamycin, an anthracycline glycoside antibiotic that inhibits the electron flow in mitochondria, also inhibits photosynthetic electron transport (PSI+PSII). The oxygen consumption curves suggest an inhibitory effect of PSII activity at very low adriamycin concentrations. Surface potential and differential scanning calorimetry measurements coupled with the use of tritiated daunomycin demonstrated that adriamycin interacts specifically with negatively charged thylakoid lipids, and induces a clustering of these negatively charged lipids in a neutral lipid matrix. These properties have made it possible to suggest a mechanism for the adriamycin-induced inhibition of mitochondrial enzymes (cytochrome c oxidase, NADH: cytochrome c oxidoreductase). We did not identify precisely the target responsible for the adriamycin effect in the thylakoid membrane, but the preliminary studies reported herein indicate evident similarities between the two inhibition mechanisms.     

Interfacial electrochemistry of cytochrome c at tin oxide,indium oxide,gold, and platinum electrodes

Cyclic voltammetry has been used to study the heterogeneous electron transfer kinetics of horse heart cytochrome c in pH 7 tris/cacodylate media at several electrode surfaces. Reversible voltammetric responses (formal heterogeneous electron transfer rate constant>10^?2 cm/s) were observed at bare gold electrodes and at tin-doped indium oxide semiconductor electrodes for certain experimental conditions. Quasireversible voltammetric responses were more typically observed at fluorine-doped tin oxide semiconductor electrodes, bare platinum electrodes, and at the indium oxide electrodes. Reaction rates at bare metal electrodes were strongly dependent on pretreatment procedures and experimental protocol. Reaction rates at metal oxide electrodes were strongly dependent on solution conditions, pretreatment procedures, and on the hydration state of the electrode surface. A general mechanistic scheme involving both interfacial electrostatic and chemical interactions is proposed for cytochrome c electrode reactions. The asymmetric distribution of surface charges on cytochrome c appears to play a dominant role in controlling electron transfer rates by its interaction with the electric field at the electrode surface. Electron transfer distances are also considered, and it is concluded that electron transfer between an electrode surface and the exposed heme edge of properly oriented cytochrome c molecules involves maximum distances of ca. 0.6–0.9 nm.     

On-line collection/concentration and determination of transition and rare-earth metals in water samples using Multi-Auto-Pret system coupled with inductively coupled plasma-atomic emission spectrometry

On-line preconcentration and determination of transition and rare-earth metals in water samples was performed using a Multi-Auto-Pret system coupled with inductively coupled plasma-atomic emission spectrometry (ICP-AES). The Multi-Auto-Pret AES system proposed here consists of three Auto-Pret systems with mini-columns that can be used for the preconcentration of trace metals sequentially or simultaneously, and can reduce analysis time to one-third and running cost of argon gas and labor. A newly synthesized chelating resin, ethylenediamine-N,N,N′-triacetate-type chitosan (EDTriA-type chitosan), was employed in the Multi-Auto-Pret system for the collection of trace metals prior to their measurement by ICP-AES. The proposed resin showed very good adsorption ability for transition and rare-earth metal ions without any interference from alkali and alkaline-earth metal ions in an acidic media. For the best result, pH 5 was adopted for the collection of metal ions. Only 5 mL of samples could be used for the determination of transition metals, while 20 mL of samples was necessary for the determination of rare-earth metals. Metal ions adsorbed on the resin were eluted using 1.5 M nitric acid, and were measured by ICP-AES. The proposed method was evaluated by the analysis of SLRS-4 river water reference materials for trace metals. Good agreement with certified and reference values was obtained for most of the metals examined; it indicates that the proposed method using the newly synthesized resin could be favorably used for the determination of transition and rare-earth metals in water samples by ICP-AES.     

Direct electrochemistry of bacterial cytochrome c551 at surface-modified gold electrodes

The direct electrochemistry of the single heme cytochrome c₅₅₁ from the bacterium Pseudomonas aeruginosa has been investigated at gold electrodes surface-modified through chemisorption of polyfunctional organic molecules. The results have been compared and contrasted with those obtained under the same conditions for the eukaryotic cytochrome c from horse heart. Both cytochromes give a quasi-reversible electrode reaction at pH 6.0 at a modified interface presenting only 4-pyridyl groups to the solution suggesting the occurrence, in both cases, of a hydrogen bonding interaction from lysine side-chains on the protein to pyridyl-nitrogens on the electrode surface. However, in contrast, gold electrodes modified by Pyridine-n-AldehydeThioSemicarbazones (n = 2, 3, 4) give electrochemistry which is strongly isomer-dependent in the case of horse heart cytochrome c but completely isomer-independent in the case of cytochrome c₅₅₁. It is suggested that interaction of the eukaryotic protein with surfaces is dominated by its lysine residues only, but that interaction of the bacterial cytochrome is through hydrogen bonding from the surface to both lysines and carboxylate groups of aspartate residues. This is supported by observation of the loss of cytochrome c₅₅₁ electrochemistry at 4-pyridyl-only modified gold at pH 9.0 compared with the good, quasi-reversible electrochemistry maintained under the same conditions at PATS-4 modified gold. It is concluded that, while the two cytochromes show many similarities with respect to their structures and functions, they have quite different interfacial electron transfer reactions, particularly at PATS-modified electrodes. This may correlate with the known large differences between the two proteins in net electrostatic charge and surface charge distribution.     

Use of ring-disc electrodes and viologens for the titration of cytochrome C and oxygen and for the study of their reduction kinetics

The use of ring-disc electrodes enable the electrogeneration of viologen cation radicals V⁺ when the disc potential is controlled in the range ?0.3 to ?0.65 and ?0.2 to ?0.45 V/NHE respectively in MV²⁺ and BV²⁺ solutions. We confirmed the occurrence of very fast reactions between V^.+ and horse-heart cytochrome c or oxygen and of slow or no reaction between V^.+ and β-nicotinamide adenine dinucleotide with xanthine oxidase, riboflavine and ferricyanide. Ring current versus dise current curves enable the titration of cytochrome c and oxygen and the estimation of the reaction rate between cytochrome c and MV^.+ [(6.2±2)10⁵M^?1 s^?1].     

Selective fluorometry of cytochrome c using glutathione-capped CdTe quantum dots in weakly basic medium

Glutathione-capped CdTe quantum dots (GSH-CdTe QDs) were synthesized in aqueous medium. Their interactions with proteins, especially three heme-containing proteins, were investigated over a broad pH range. At 6.0?<?pH?<?8.0, the fluorescence of the GSH-CdTe QDs can be effectively quenched by cytochrome c (Cyt. c) and hemoglobin, respectively. At pH?>?8.0, only cytochrome c quenched the fluorescence of the GSH-CdTe QDs, and no significant fluorescence changes were observed for hemoglobin or other proteins. Based on the distinct fluorescence response, a novel method has been developed for the selective determination of cytochrome c using GSH-CdTe QDs as the fluorescence probe at pH 9.0. Under optimum conditions, the linear range of the calibration curve is from 3.2?×?10^?8 to 2.4?×?10^?6?mol L^?1 and the detection limit is 3.0?×?10^?9?mol L^?1. The method has been applied to the determination of cytochrome c in three synthetic and real samples, and satisfactory results were obtained with QDs through selecting the proper pH value.     

Reagentless lactate electrodes based on electrocatalytic oxidation of flavocytochrome b2

Flavocytochrome b₂ (L-lactate :cytochrome c reductase, E.C. 1.1.2.3) from Hansenula anomala was entrapped on the surface of electrodes modified with various kinds of carbon black. The electrocatalytic oxidation of a reduced enzyme by the electroactive surface groups of carbon black enables this enzyme electrode to be used for the determination of lactate. The electrodes operate at ?0.2 to ?0.1 V vs. SCE (pH 7.0), which is low enough to avoid interference from ascorbic acid. Linear calibration graphs up to 0.5 mM lactate were obtained. Electrochemical measurements of lactate in human blood plasma and cell culture fluids showed good agreement with the results of spectrophotometric measurements.     

Site-Directed Mutagenesis of Cytochrome P450 2D6 and 2C19 Enzymes: Expression and Spectral Characterization of Naturally Occurring Allelic Variants

Genetic polymorphism of the cytochrome P450 (CYP) genes particularly affects CYP2D6 and CYP2C19 to a functionally relevant extent, and it is therefore crucial to elucidate the enzyme kinetic and molecular basis for altered catalytic activity of these allelic variants. This study explored the expression and function of the reported alleles CYP2D6*2, CYP2D6*10, CYP2D6*17, CYP2C19*23, CYP2C19*24, and CYP2C19*25 with respect to gene polymorphisms. Site-directed mutagenesis (SDM) was carried out to generate these six alleles. After DNA sequencing, the CYP2D6 and CYP2C19 wild types alongside with their alleles were each independently co-expressed with NADPH-CYP oxidoreductase (OxR) in Escherichia coli. The expressed proteins were analyzed using Western blotting, reduced carbon monoxide (CO) difference spectral scanning, and cytochrome c reductase assay. Results from Western blot revealed the presence of all CYP wild-type and allelic proteins in E. coli membrane fractions. The reduced CO difference spectra scanning presented the distinct peak of absorbance at 450 nm, and the cytochrome c reductase assay has confirmed that spectrally active OxR was expressed in each protein preparation. As a conclusion, the results obtained from this study have proven the CYP variants to be immunoreactive and spectrally active and are suitable for use to examine biotransformation and interaction mechanism of the enzymes.     

Solid-phase extraction of lead(II) ions using multiwalled carbon nanotubes grafted with tris(2-aminoethyl)amine

Multiwalled carbon nanotubes were grafted with tris(2-aminoethyl)amine (MWCNTs-TAA) and employed for solid phase extraction and preconcentration of trace lead ions prior to its determination by inductively coupled plasma optical emission spectrometry. The material was characterized by FT-IR and Raman spectroscopy, thermosgravimetric and elemental analysis. The effects of pH value, shaking time, sample volume, elution conditions and potentially interfering ions were investigated. Under the optimum conditions, the maximum adsorption capacity is 38?mg?g^?1 of Pb(II), the detection limit is 0.32?ng?mL^?1, the enrichment factor is 60, and the relative standard deviation is 3.5% (n?=?6). The method has been applied to the preconcentration of trace amounts of Pb(II) in environmental water samples with satisfactory results.

Synthesis, Characterization of Heterodinuclear Co-Cu Complex and Its Electrocatalytic Activity towards 02 Reduction： Implications for Cytochrome c Oxidase Active Site Modeling

A new dinudeating ligand consisting of a tetraphanylporphyrin derivative covalently linked with tris(2-benzimidazylmethyl)-amine and its homodinudear Co-Co and heterodinnelear Co-Cu complexes were synthesized and spectroscopically character-ized. The heterobimetallie cobalt-copper complex bearing three benzimidazole ligands for copper, as cytochrome c oxidase ac-tive site model, was applied to the surface of glassy carbon elec-trode to show electrocatalytie activity for O2 reduction in aque-ous solution at an addity level dose to physiological pH value.The kinetic parameters of this electrocatalytic process were ob-tained.