We studied in this work the performance of the new ultrasonic multiprobe in terms of throughput, handling and robustness. The study was conducted using the multiprobe to speed two different proteomics workflows. The "classic" method relaying on overnight protein digestion (12h), was used as the standard procedure. This work clearly shows the importance of testing variables such as ultrasonic amplitude and ultrasonic time when adapting an ultrasonic-based treatment to a new ultrasonic device. The results here presented also shown and confirm the advantage of speed up sample treatment workflows with the aid of ultrasonic energy in combination with a 96-well plate. The methods compared were similar in terms of robustness, but the desalting free method was the fastest, requiring only 2 min/sample for completion. In addition it was also the simplest in terms of handling, since no desalting step was needed. The following standard proteins were successfully identified using the methods studied: bovine serum albumin, alpha-lactalbumin, ovalbumin, carbonic anhydrase, fructose-bisphosphate aldolase A, catalase, chymotrypsinogen A. As case study, the identification of the protein Split-Soret cytochrome c from D. desulfuricans ATCC 27774 was carried out.

We report in this work on the robustness of ultrasonic energy as a tool to speed the isotopic labeling of proteins using the (18)O-decoupling procedure. The first part of the decoupling procedure, comprising protein denaturation, reduction, alkylation and digestion, is done in 8 min under the effects of an ultrasonic field whilst the second part, the isotopic labeling, was assayed with and without the use of ultrasonic energy. Our results clearly demonstrate that the (18)O-isotopic labeling in a decoupling procedure cannot be accelerated using an ultrasonic field.

We report in this work on the robustness of ultrasonic energy as a tool to speed the isotopic labeling of proteins using the (18)O-decoupling procedure. The first part of the decoupling procedure, comprising protein denaturation, reduction, alkylation and digestion, is done in 8 min under the effects of an ultrasonic field whilst the second part, the isotopic labeling, was assayed with and without the use of ultrasonic energy. Our results clearly demonstrate that the (18)O-isotopic labeling in a decoupling procedure cannot be accelerated using an ultrasonic field.

A conductometric biosensor for nitrite detection was developed using cytochrome c nitrite reductase (ccNiR) extracted from Desulfovibrio desulfuricans ATCC 27774 cells immobilized on a planar interdigitated electrode by cross-linking with saturated glutaraldehyde (GA) vapour in the presence of bovine serum albumin, methyl viologen (MV), Nafion, and glycerol. The configuration parameters for this biosensor, including the enzyme concentration, ccNiR/BSA ratio, MV concentration, and Nafion concentration, were optimized. Various experimental parameters, such as sodium dithionite added, working buffer solution, and temperature, were investigated with regard to their effect on the conductance response of the biosensor to nitrite. Under the optimum conditions at room temperature (about 25 degrees C), the conductometric biosensor showed a fast response to nitrite (about 10s) with a linear range of 0.2-120 microM, a sensitivity of 0.194 microS/microM [NO(2)(-)], and a detection limit of 0.05 microM. The biosensor also showed satisfactory reproducibility (relative standard deviation of 6%, n=5). The apparent Michaelis-Menten constant (K(M,app)) was 338 microM. When stored in potassium phosphate buffer (100mM, pH 7.6) at 4 degrees C, the biosensor showed good stability over 1 month. No obvious interference from other ionic species familiar in natural waters was detected. The application experiments show that the biosensor is suitable for use in real water samples.

The characterization of a novel Mo-Fe protein (MorP) associated with a system that responds to Mo in Desulfovibrio alaskensis is reported. Biochemical characterization shows that MorP is a periplasmic homomultimer of high molecular weight (260 +/- 13 kDa) consisting of 16-18 monomers of 15321.1 +/- 0.5 Da. The UV/visible absorption spectrum of the as-isolated protein shows absorption peaks around 280, 320, and 570 nm with extinction coefficients of 18700, 12800, and 5000 M(-1) cm(-1), respectively. Metal content, EXAFS data and DFT calculations support the presence of a Mo-2S-[2Fe-2S]-2S-Mo cluster never reported before. Analysis of the available genomes from Desulfovibrio species shows that the MorP encoding gene is located downstream of a sensor and a regulator gene. This type of gene arrangement, called two component system, is used by the cell to regulate diverse physiological processes in response to changes in environmental conditions. Increase of both gene expression and protein production was observed when cells were cultured in the presence of 45 microM molybdenum. Involvement of this system in Mo tolerance of sulfate reducing bacteria is proposed.

Membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617 can be solubilized in either of two ways that will ultimately determine the presence or absence of the small (Iota) subunit. The enzyme complex (NarGHI) is composed of three subunits with molecular masses of 130, 65, and 20 kDa. This enzyme contains approximately 14 Fe, 0.8 Mo, and 1.3 molybdopterin guanine dinucleotides per enzyme molecule. Curiously, one heme b and 0.4 heme c per enzyme molecule have been detected. These hemes were potentiometrically characterized by optical spectroscopy at pH 7.6 and two noninteracting species were identified with respective midpoint potentials at Em=+197 mV (heme c) and -4.5 mV (heme b). Variable-temperature (4-120 K) X-band electron paramagnetic resonance (EPR) studies performed on both as-isolated and dithionite-reduced nitrate reductase showed, respectively, an EPR signal characteristic of a [3Fe-4S]+ cluster and overlapping signals associated with at least three types of [4Fe-4S]+ centers. EPR of the as-isolated enzyme shows two distinct pH-dependent Mo(V) signals with hyperfine coupling to a solvent-exchangeable proton. These signals, called "low-pH" and "high-pH," changed to a pH-independent Mo(V) signal upon nitrate or nitrite addition. Nitrate addition to dithionite-reduced samples at pH 6 and 7.6 yields some of the EPR signals described above and a new rhombic signal that has no hyperfine structure. The relationship between the distinct EPR-active Mo(V) species and their plausible structures is discussed on the basis of the structural information available to date for closely related membrane-bound nitrate reductases.

Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterised in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the "LID" domain. The sequence 129Cys-X5-His-X15-Cys-X2-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain.

The influence of the protein staining used to visualize protein bands, after in-gel protein separation, for the correct identification of proteins by peptide mass fingerprint (PMF) after application of the ultrasonic in-gel protein protocol was studied. Coomassie brilliant blue and silver nitrate, both visible stains, and the fluorescent dyes Sypro Red and Sypro Orange were evaluated. Results obtained after comparison with the overnight in-gel protocol showed that good results, in terms of protein sequence coverage and number of peptides matched, can be obtained with anyone of the four stains studied. Two minutes of enzymatic digestion time was enough for proteins stained with coomassie blue, while 4 min was necessary when silver or Sypro stainings were employed in order to reach equivalent results to those obtained for the overnigh in-gel protein protocol. For the silver nitrate stain, the concentration of silver present in the staining solution must be 0.09% (w/v) to minimize background in the MALDI mass spectra.

A new clean fast (8 min) method for in-solution protein digestion Without detergent or urea for protein identification by peptide mass fingerprint and mass spectrometry-based techniques is Proposed. The new method avoids the use of time consuming desalting procedures entailing the following four steps done under the effect of an ultrasonic field provided by a sonoreactor: denaturation (1 min) in a mixed Solution of water:acetonitrile 1/1 (v/v): protein reduction (1 min); protein alkylation (1 min); and protein digestion (5 min). Five Proteins with masses comprised between 14.4 kDa and 97 kDa and the protein splitsoret cytochrome c from D. desulfuricans ATCC27774, Were Successfully identified with this procedure. No differences were found in the sequence coverage or in the number of peptides matched when the new clean method was compared to another one using urea. Twofold better signal-to-noise ratios were obtained in the MALDI spectra from protein samples prepared with the new method when comparing it with a method using urea. The new digestion method avoids the need to remove salt content and increases throughput (six samples at once) while reducing sample loss and contamination from sample handling. (C) 2008 Elsevier B.V. All rights reserved.

Sulphate-reducing bacteria have a wide variety of periplasmic cytochromes involved in electron transfer from the periplasm to the cytoplasm. HmcA is a high molecular mass cytochrome of 550 amino acid residues that harbours 16 c-type heme groups. We report the crystal structure of HmcA isolated from the periplasm of Desulfovibrio gigas. Crystals were grown. using polyethylene glycol 8K and zinc acetate, and diffracted beyond 2.1 angstrom resolution. A multiple-wavelength anomalous dispersion experiment at the iron absorption edge enabled us to obtain good-quality phases for structure solution and model building. DgHmcA has a V-shape architecture, already observed in HmcA isolated from Desulfovibrio vulgaris Hildenborough. The presence of an oligosaccharide molecule covalently bound to an Asn residue was observed in the electron density maps of DgHmcA and confirmed by mass spectrometry. Three modified monosaccharides appear at the highly hydrophobic vertex, possibly acting as an anchor of the protein to the cytoplasmic membrane. (c) 2007 Elsevier Ltd. All rights reserved.

Cytochrome c nitrite reductase is a multicenter enzyme that uses a five-coordinated heme to perform the six-electron reduction of nitrite to ammonium. In the sulfate reducing bacterium Desulfovibrio desulfuricans ATCC 27774, the enzyme is purified as a NrfA2NrfH complex that houses 14 hemes. The number of closely-spaced hemes in this enzyme and the magnetic interactions between them make it very difficult to study the active site by using traditional spectroscopic approaches such as EPR or UV-Vis. Here, we use both catalytic and non-catalytic protein film voltammetry to simply and unambiguously determine the reduction potential of the catalytic heme over a wide range of pH and we demonstrate that proton transfer is coupled to electron transfer at the active site.

Rapid identification of proteins is of primary importance for the analytical community. Protein-biomarker discovery for medical diagnostics or pharmacological purposes is becoming one of the hottest research topics. Moreover, rapid identification of proteins can help unambiguous bacterial and virus detection. In addition, the fast identification of bacteria can be used to beat bioterrorism. As a consequence, new analytical methodologies have emerged recently with the aim of making protein analysis as fast and as confident as possible. In this article, we critically review the new trends in sample treatment for protein identification and comment on the prospects for the future in this promising analytical area. (c) 2006 Elsevier Ltd. All rights reserved.

Ferredoxin (Fd) and ferredoxin-NADP(+)-reductase (FNR) are two terminal physiological partners of the photosynthetic electron transport chain. Based on a nuclear magnetic resonance (NMR)-restrained-docking approach, two alternative structural models of the Fd-FNR complex in the presence of NADP+ are proposed. The protein docking simulations were performed with the software BiGGER. NMR titration revealed a 1:1 stoichiometry for the complex and allowed the mapping of the interacting residues at the surface of Fd. The NMR chemical shifts were encoded into distance constraints and used with theoretically calculated electronic coupling between the redox cofactors to propose experimentally validated docked complexes.

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