Purification and characterization of the recombinant Na⁺-translocating NADH : quinone oxidoreductase from vibrio cholerae

The nqr operon from Vibrio cholerae, encoding the entire six-subunit, membrane-associated, Na+-translocating NADH:quinone oxidoreductase (Na+-NQR), was cloned under the regulation of the PBAD promoter. The enzyme was successfully expressed in V. cholerae. To facilitate molecular genetics studies of this sodium-pumping enzyme, a host strain of V. cholerae was constructed in which the genomic copy of the nqr operon was deleted. By using a vector containing a six-histidine tag on the carboxy terminus of the NqrF subunit, the last subunit in the operon, the recombinant enzyme was readily purified by affinity chromatography in a highly active form from detergent-solubilized membranes of V. cholerae. The recombinant enzyme has a high specific activity in the presence of sodium. NADH consumption was assessed at a turnover number of 720 electrons per second. When purified using dodecyl maltoside (DM), the isolated enzyme contains approximately one bound ubiquinone, whereas if the detergent LDAO is used instead, the quinone content of the isolated enzyme is negligible. Furthermore, the recombinant enzyme, purified with DM, has a relatively low rate of reaction with O2 (10-20 s(-1)). In steady state turnover, the isolated, recombinant enzyme exhibits up to 5-fold stimulation by sodium and functions as a primary sodium pump, as reported previously for Na+-NQR from other bacterial sources. When reconstituted into liposomes, the recombinant Na+-NQR generates a sodium gradient and a Deltapsi across the membrane. SDS-PAGE resolves all six subunits, two of which, NqrB and NqrC, contain covalently bound flavin. A redox titration of the enzyme, monitored by UV-visible spectroscopy, reveals three n = 2 redox centers and one n = 1 redox center, for which the presence of three flavins and a 2Fe-2S center can account. The V. cholerae Na+-NQR is well-suited for structural studies and for the use of molecular genetics techniques in addressing the mechanism by which NADH oxidation is coupled to the pumping of Na+ across the membrane

Highly Sensitive and Selective Colorimetric Sensors for Uranyl (UO22+): Development and Comparison of Labeled and Label-Free DNAzyme-Gold Nanoparticle Systems

Colorimetric uranium sensors based on uranyl (UO(2)(2+)) specific DNAzyme and gold nanoparticles (AuNP) have been developed and demonstrated using both labeled and label-free methods. In the labeled method, a uranyl-specific DNAzyme was attached to AuNP, forming purple aggregates. The presence of uranyl induced disassembly of the DNAzyme functionalized AuNP aggregates, resulting in red individual AuNPs. Once assembled, such a “turn-on” sensor is highly stable and worked in a single step at room temperature and had detection limit of 50 nM after 30 min. of reaction time. The label-free method, on the other hand, utilizes the different adsorption properties of single stranded and double stranded DNA on AuNPs, which affects the stability of AuNPs in the presence of NaCl. The presence of uranyl resulted in cleavage of substrate by DNAzyme, releasing a single stranded DNA that can be adsorbed on AuNPs and protect them from aggregation. Taking advantage of this phenomenon, a “turn-off” sensor was developed, which is easy to control through reaction quenching, and has 1 nM detection limit after 6 min. of reaction at room temperature. Both sensors have excellent selectivity over other metal ions and have detection limits below the maximum contamination level of 130 nM for UO(2)(2+) in drinking water defined by the US Environmental Protection Agency (EPA). The study represents the first direct systematic comparison of these two types of sensor methods using the same DNAzyme and AuNPs, making it possible to reveal advantages, disadvantages, versatility, and limitations and potential applications of each method. The results obtained not only allow practical sensing application for uranyl, but also serve as a guide for choosing different method for designing colorimetric sensors for other targets

Characterization of metal ion-nucleic acid interactions in solution

Metal ions are inextricably involved with nucleic acids due to their polyanionic nature. In order to understand the structure and function of RNAs and DNAs, one needs to have detailed pictures on the structural, thermodynamic, and kinetic properties of metal ion interactions with these biomacromolecules. In this review we first compile the physicochemical properties of metal ions found and used in combination with nucleic acids in solution. The main part then describes the various methods developed over the past decades to investigate metal ion binding by nucleic acids in solution. This includes for example hydrolytic and radical cleavage experiments, mutational approaches, as well as kinetic isotope effects. In addition, spectroscopic techniques like EPR, lanthanide(III) luminescence, IR and Raman as well as various NMR methods are summarized. Aside from gaining knowledge about the thermodynamic properties on the metal ion-nucleic acid interactions, especially NMR can be used to extract information on the kinetics of ligand exchange rates of the metal ions applied. The final section deals with the influence of anions, buffers, and the solvent permittivity on the binding equilibria between metal ions and nucleic acids. Little is known on some of these aspects, but it is clear that these three factors have a large influence on the interaction between metal ions and nucleic acids