Electrochemical control of protein monolayers at indium tin oxide surfaces for the reagentless optical biosensing of nitric oxide

Cytochrome c has been immobilized onto functionalized, optically transparent indium tin oxide (ITO) electrodes by covalent and electrostatic techniques. Covalent immobilization was achieved by the formation of a disulfide bond between N-succinimidyl 3-(2-pyridyldithio)propionate-(SPDP-) modified cytochrome c and SPDP-silanized ITO. Additionally, ITO electrodes have been modified with the bifunctional reagent 1,12-dodecanedicarboxylic acid (DDCA), resulting in formation of a carboxylic acid-terminated monolayer. Covalent protein attachment to the DDCA-functionalized ITO was achieved with the cross-linker 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride. Electrostatic attachment of the protein involved ion-pair and hydrogen-bond interactions between the terminating carboxylic acid groups of the DDCA-functionalized ITO and the primary amine groups of the lysine residues of cytochrome c. The electrostatic interaction between the cytochrome c and the functionalized ITO resulted in greater rotational mobility of the protein at the electrode surface, leading to ca. 63% electroactivity, as compared to ca. 41% electroactivity for the covalently immobilized protein. The redox state of the electrostatically bound cytochrome c monolayers could be electrochemically switched between ferric and ferrous forms. Electrochemical control of the bound protein was used to regenerate the biosensing surface following binding of nitric oxide (NO). Ligation of NO with the cytochrome c was monitored by measurement of the change of absorbance intensity at 416 nm. Through application of a negative potential, the cytochrome c was reduced from the ferric to the ferrous form, which led to the removal of the ligated NO. Application of a positive potential regenerated the ferric cytochrome c, enabling multiple repeat measurements of NO. Such electrochemical control of proteins immobilized on transparent electrodes enables the optical biosensing of analyte targets without recourse to exogenous reagents.     

Electron affinity of the guanine-cytosine base pair and structural perturbations upon anion formation

The adiabatic electron affinity (AEA) for the Watson-Crick guanine-cytosine (GC) DNA base pair is predicted using a range of density functional methods with double- and triple-zeta plus polarization plus diffuse (DZP++ and TZ2P++) basis sets in an effort to bracket the true electron affinity. The methods used have been calibrated against a comprehensive tabulation of experimental electron affinities (Chem.Rev. 2002, 102, 231). Optimized structures for GC and the GC anion are compared to the neutral and anionic forms of the individual bases as well as Rich's 1976 X-ray structure for sodium guanylyl-3',5'-cytidine nonahydrate, GpC.9H(2)O. Structural distortions and natural population (NPA) charge distributions of the GC anion indicate that the unpaired electron is localized primarily on the cytosine moiety. Unlike treatments using second-order perturbation theory (MP2), density functional theory consistently predicts a substantial positive adiabatic electron affinity for the GC pair (e.g., TZ2P++/B3LYP: +0.48 eV). The stabilization of C(-) via three hydrogen bonds to guanine is sufficient to facilitate adiabatic binding of an electron to GC and is also consistent with the positive experimental electron affinities obtained by photoelectron spectroscopy of cytosine anions incrementally microsolvated with water molecules. The pairing (dissociation) energy for GC(-) (35.6 kcal/mol) is determined with inclusion of electron correlation and shows the anion to have greater thermodynamic stability; the pairing energy for neutral GC (TZ2P++/B3LYP 23.9 kcal/mol) compares favorably to previous MP2/6-31G (23.4 kcal/mol) results and a debated experiment (21.0 kcal/mol).     

Incoherent neutron quasi-elastic scattering studies of the anisotropic self-diffusion in nematic and smectic a phases of ethyl 4-(4′acetoxy benzylidene) aminocinnamate (EABAC)

Incoherent quasi-elastic neutron scattering spectra have been measured down to low scattering vectors (Q >) 0.14 Å^?1) on magnetically aligned specimens of the nematic and smectic A phases of EABAC. Data were obtained for Q || n and Q⊥n where n denotes the direction of the unique axis) giving the diffusion coefficients D_|| and D_⊥ for the two phases as follows (in units of 10^?7 cm² s^?1); nematic: D_|| = 15.8 ± 2.0, D_⊥ = 12.3 ± 0.8; smectic A: D_|| = 4.3 ± 0.4, D_⊥ = 4.9 ± 0.8. The anisotropy is reversed between the phases and the molecules are more mobile in the nematic phase as expected. In the smectic A experiments with Q || n an apparent inconsistency between low and high Q results is interpreted as evidence for the existence of some slow motion other than translational diffusion which requires further investigation. Preliminary measurements were made to explore the application of the “fixed window” method for determining the temperature dependence of the diffusion coefficients.     

Reductive activation of nitrate reductases

Protein film voltammetry of Paracoccus pantotrophus respiratory nitrate reductase (NarGH) and Synechococcus elongatus assimilatory nitrate reductase (NarB) shows that reductive activation of these enzymes may be required before steady state catalysis is observed. For NarGH complementary spectroscopic studies suggest a structural context for the activation. Catalytic protein film voltammetry at a range of temperatures has allowed quantitation of the activation energies for nitrate reduction. For NarGH with an operating potential of ca. 0.05 V the activation energy of ca. 35 kJ mol-1 is over twice that measured for NarB whose operating potential is ca. -0.35 V.     

Identification of reactive dyes in spent dyebaths and wastewater by capillary electrophoresis-mass spectrometry

Capillary electrophoresis with diode array detection and mass spectrometry combined with solid-phase extraction were employed for the identification of reactive vinylsulfone and chlorotriazine dyes and their hydrolysis products in spent dyebaths and raw and treated wastewater. Recoveries of dyes from treated wastewater as their tetrabutylammonium ion-pairs using C18 reversed-phase cartridges ranged from 81 to 121%. Detection limits in sewage effluent of the different dyes and hydrolysis products ranged from 23 to 42 microg/l. The method was successfully applied to the detection of the hydrolysis products of five reactive dyes in influents and effluents of a municipal wastewater treatment plant receiving dyehouse effluents.     

Synthesis and antibacterial activity of 7-ethyl-3-methyl-4,7-dihydro-4-oxoisothiazolo[5,4-b]pyridine-5-carboxylic acid

The synthesis of the quinolone, 7-ethyl-3-methyl-4,7-dihydro-4-oxoisothiazolo[5,4-b]pyridine-5-carboxylic acid 4 was accomplished utilizing the Gould-Jacobs dependent route. The compound had very weak in vitro activity as compared to nalidixic acid versus E. coli, P. aeruginosa, K. pnuemoniae, S. aureus and P. mirabilis.     

The first structural and spectroscopic characterization of a neptunyl polyoxometalate complex

The reaction between PW9O349- and NpO2+ has yielded the first structurally characterized neptunyl(V) polyoxometalate complex, [Na2(NpO2)2(A-PW9O34)2]14-. This complex is isostructural with the uranyl(VI) analogue, and there is also spectroscopic evidence for its existence in solution. The complex is readily extracted into toluene, and this may have significance in the sequestering and/or separation of the neptunyl ion in terms of nuclear waste management.     

Electronic structure, vibrational stability, and predicted infrared-Raman spectra of the As20, As @ Ni12, and As @ Ni12 @ As20 clusters

Recently an inorganic fullerine-like [As@Ni(12)@As(20)](3-) onion with near-perfect icosahedral symmetry in the crystalline phase was reported [M. J. Moses, J. C. Fettinger, and B. W. Eichhorn, Science 300, 778 (2003)]. This paper presents a detailed computational study in the framework of density functional theory on various aspects of this molecule. The electronic structure of the As@Ni(12)@As(20) is investigated in its neutral as well as -3 charged state together with its subunits As(20) and As@Ni(12) by the all electron linear combination of Gaussian-type orbitals method. The bonding is studied by examining the integrated charge within atomic sphere, the electron localization function, changes in the electron density distribution, and from vibrational modes. We find that strong covalent As-As bonds seen in isolated As(20) become weaker in the As@Ni(12)@As(20) and strong covalent As-Ni bonds are formed. The structural stability of all four clusters is examined by analyzing the energetics and by calculating the vibrational frequencies. Further, the infrared and Raman spectra is predicted for both the neutral and charged As@Ni(12)@As(20) clusters. Finally, the energy barrier for removal of a single arsenic atom is calculated for the neutral As@Ni(12)@As(20) cluster.     

The complexity of catalysis: origins of enantio- and diastereocontrol in sulfur ylide mediated epoxidation reactions

The reaction of chiral sulfur ylides with aldehydes and ketones has emerged as a useful asymmetric process for the synthesis of epoxides. Processes employing either catalytic or stoichiometric amounts of sulfides have been developed. Although a large number of chiral sulfur ylides have been tested in the epoxidation process, only a few have delivered high diastereo- and enantio- selectivity. This review examines the factors that influence stereocontrol (steric hindrance of the sulfide, ylide conformation, ylide face selectivity, reversibility of betaine formation, solvent, and metal salts). This analysis leads to the conclusion that high reversibility in betaine formation leads to high diastereoselectivity but low enantioselectivity, and non-reversible betaine formation leads to low diastereoselectivity and high enantioselectivity (provided that other criteria are met). To achieve both high diastereoselectivity and high enantioselectivity simultaneously, requires non-reversible formation of the anti-betaine and reversible formation of the syn-betaine. Thus, factors that influence the degree of reversibility in betaine formation are critically important since with subtle changes in reaction conditions (solvent, temperature, metal ions) both high enantio- and diastereoselectivity can often be achieved.     

Average peptide score: a useful parameter for identification of proteins derived from database searches of liquid chromatography/tandem mass spectrometry data

The quantity and variable quality of data that can be generated from liquid chromatography (LC)/mass spectrometry (MS)-based proteomics analyses creates many challenges in interpreting the spectra in terms of the actual proteins in a complex sample. In spite of improvements in algorithms that match putative peptide sequences to MS/MS spectra, the assembly of these lists of possible or probable peptides into a 'correct' set of proteins is still problematic. We have observed a trend in a simple relationship, derived from standard database search outputs, which can be useful in assessing the quality of a MS/MS-based protein identification. Specifically, the ratio of the protein score and number of non-redundant peptides, or average peptide score (APS), can facilitate initial filtering of database search results in addition to providing a useful measure of confidence for the proteins identified. This parameter has been applied to results from the analysis of multi-protein complexes derived from pull-down experiments analyzed using a two-dimensional LC/MS/MS workflow. In particular, the complex list of protein identifications derived from a drug affinity pull-down with immobilized ampicillin and an E. coli lysate was greatly simplified by applying the APS as a filter, allowing for facile identification of the penicillin-binding proteins known to interact with ampicillin. Furthermore, an APS threshold can be used for any data sets derived from electrospray ionization (ESI)- or matrix-assisted laser desorption/ionization (MALDI)-MS/MS experiments and is also not specific to any database search program.