Formate oxidase, an enzyme of the glucose-methanol-choline oxidoreductase family, has a His-Arg pair and 8-formyl-FAD at the catalytic site.

Formate oxidase of Aspergillus oryzae RIB40 contains an 8-replaced FAD with molecular mass of 799 as cofactor. The ¹H-NMR spectrum of the cofactor fraction obtained from the enzyme indicated that the 8-replaced FAD in the fraction was 8-formyl-FAD, present in open form and hemiacetal form. The oxidation-reduction potentials of the open and hemiacetal forms were estimated by cyclic voltammetry to be -47 and -177 mV vs. Normal Hydrogen Electrode respectively. The structure of the enzyme was constructed using diffraction data to 2.24 Å resolution collected from a crystal of the enzyme. His511 and Arg554 were situated close to the pyrimidine part of the isoalloxazine ring of 8-formyl-FAD in open form. The enzyme had 8-formyl-FAD, the oxidation potential of which was approximately 160 mV more positive than that of FAD, and the His-Arg pair at the catalytic site, unlike the other enzymes belonging to the glucose-methanol-choline oxidoreductase family.

1-Butyl-3-methylimidazolium hexafluorophosphate ionic liquid as a new solvent for the determination of lead(II) and cadmium(II) by anodic stripping voltammetry after extraction of the iodide complexes.

The use of 1-butyl-3-methylimidazolium hexafluorophosphate (BMIM x PF(6)) as a solvent for the anodic stripping analysis of Pb(II) and Cd(II) after extraction of the iodide complexes has been investigated. This method is based on the extraction of the metal iodide complexes into BMIM x PF(6), followed by differential-pulse anodic stripping voltammetry with an in-situ plated bismuth film electrode onto an edge-plane type of pyrolytic graphite substrate. When tetra-n-propylammonium iodide at a concentration of 0.20 mol L(-1) was used as an extractant and a supporting electrolyte, the reduced lead and cadmium produced well-defined anodic peaks at -414 and -736 mV vs. Ag/AgCl, respectively. The peak currents for Pb(II) and Cd(II) were directly proportional to the initial metal concentration in the ranges of 0.01-0.50 microg mL(-1) and 0.05-1.0 microg mL(-1) under the optimized conditions. A detection limit (S/N = 3) of 0.001 microg mL(-1) Pb(II) was obtained with a volume ratio (V(aq)/V(BMIM x PF(6))) of 2.0 at 300 s deposition time. The relative standard deviation was 3.2% on replicate runs (n = 10) for the determinations of 0.20 microg mL(-1) Pb(II).

Determination of selenium(IV) by catalytic stripping voltammetry with an in situ plated bismuth-film electrode.

A very sensitive and simple method is presented for the determination of Se(IV) by Osteryang square-wave cathodic stripping voltammery (OSWCSV). The method is based on the reduction of Se(IV) with Bi(III) onto an edge-plane type of pyrolytic graphite substrate, followed by a cathodic potential scan. OSWCSV studies indicate that the reduced selenium produced a distinct catalytic hydrogen wave at -1150 mV vs. Ag/AgCl. The peak height of the catalytic hydrogen wave was directly proportional to the initial Se(IV) concentration in the ranges of 0.1 - 1.0 and 1.0 - 20.0 microg L(-1) (correlation coefficients 0.9800 and 0.9901, respectively) when the optimized parameters were used. A 3sigma detection limit of 0.025 microg L(-1)0 Se(IV) was obtained at 30 s deposition time. The relative standard deviation was 4.0% on replicate runs (n = 12) for the determinations of 0.10 microg L(-1) Se(IV). Analytical results of natural water samples demonstrate that the proposed method is applicable to speciation analysis of Se(IV) and Se(VI).

Cathodic stripping voltammetric determination of As(III) with in situ plated bismuth-film electrode using the catalytic hydrogen wave.

A highly sensitive method has been developed for the determination of trace As(III) by a square wave cathodic stripping voltammetry employing in situ plated bismuth-film on edge-plane graphite substrate as working electrode. The presence of As(III) enhanced a cathodic peak corresponding to the catalytic hydrogen wave due to Se(IV) at about -1150 mV. Linear calibration curves for As(III) determination were obtained over the concentration ranges of 0.01-1.0 microg L(-1) and 1.0-12.0 microg L(-1) at deposition times of 30 s and 10 s, respectively. The detection limit (3sigma) was estimated to be as low as 0.7 ng L(-1) As(III) at 30 s deposition time. The optimum experimental parameters and probable interference from foreign ions and organic compounds were investigated. This proposed method could be applied to analyses of certified reference material, synthetic and natural water samples.

Determination of cadmium, zinc, nickel and cobalt in tobacco by reversed-phase high-performance liquid chromatography with 2-(8-quinolylazo)-4,5-diphenylimidazole as a chelating reagent.

A sensitive and selective method has been developed for the simultaneous determination of cadmium, zinc, nickel and cobalt. The method is based on the chelation of metal ions with 2-(8-quinolylazo)-4,5-diphenylimidazole (QAI) and the subsequent reversed-phase (RP) high-performance liquid chromatographic separation and spectrophotometric detection of the metal chelates. The chelates were separated on an RP column with acetonitrile-water containing ethylenediamine tetraacetic acid and sodium acetate (pH 7.5). Though Zn(II) and Cd(II) chelates with azo compounds were generally labile in the RP column, these chelates with QAI were successfully detected. When analyses were carried out at 575 nm and at 0.001 absorbance unit full scale, the peak height calibration curves were linear up to 2.0 ng for Cd(II), 2.4 ng for Zn(II), 0.14 ng for Ni(II) and 0.72 ng for Co(II) in 100-microL injections, respectively; the detection limits (3sigma, three times of the standard deviation for the blank signal) for Cd(II), Zn(II), Ni(II) and Co(II) were 4.8, 24, 2.4 and 7.2 pg in 100 microL of injected solution, respectively. The proposed method was successfully applied to the analysis of tobacco without any preliminary concentration or separation.

Purification and characterization of an aldehyde oxidase from Pseudomonas sp. KY 4690.

An aldehyde oxidase, which oxidizes various aliphatic and aromatic aldehydes using O(2) as an electron acceptor, was purified from the cell-free extracts of Pseudomonas sp. KY 4690, a soil isolate, to an electrophoretically homogeneous state. The purified enzyme had a molecular mass of 132 kDa and consisted of three non-identical subunits with molecular masses of 88, 39, and 18 kDa. The absorption spectrum of the purified enzyme showed characteristics of an enzyme belonging to the xanthine oxidase family. The enzyme contained 0.89 mol of flavin adenine dinucleotide, 1.0 mol of molybdenum, 3.6 mol of acid-labile sulfur, and 0.90 mol of 5'-CMP per mol of enzyme protein, on the basis of its molecular mass of 145 kDa. Molecular oxygen served as the sole electron acceptor. These results suggest that aldehyde oxidase from Pseudomonas sp. KY 4690 is a new member of the xanthine oxidase family and might contain 1 mol of molybdenum-molybdpterin-cytosine dinucleotide, 1 mol of flavin adenine dinucleotide, and 2 mol of [2Fe-2S] clusters per mol of enzyme protein. The enzyme showed high reaction rates toward various aliphatic and aromatic aldehydes and high thermostability.