Characterization of solution species relevant to the peroxidase-NADH biochemical oscillator

Investigations of chemical behavior of several species essential for understanding the peroxidase/NADH oscillatory system are presented, along with development of instrumentation for superoxide radical and hydrogen peroxide detection and experimental characterization of the oscillatory system.;The dual enzyme-based amperometric sensor was applied to detection of superoxide radical and hydrogen peroxide in the peroxidase/NADH oscillator. Both species were detected at nanomolar concentrations. Concentration-time profiles for superoxide and hydrogen peroxide closely resembled qualitative and quantitative model predictions except during periods of large-d(O 2) /dt. Introduction of the sensor did not cause perturbation of the system.;The mechanism and kinetics of interaction of NADH with iron ions in pH 5.1 0.1 M acetic acid/sodium acetate buffer with and with the solution/stainless steel interface were studied. A possible mutual influence of NADWacetate buffer solution and Type 316 stainless steel was investigated. The mechanism of stainless steel corrosion inhibition by NADH was also suggested. Fast complexation of ferric and ferrous ions with NADH occurred with rate constant kcompl =4.0.109+/-0.2.109 M --1 sec--1 The composition of the product complex is [Fe-(NADH)2] for both Fe2+ and Fe 3+. A previously unreported complex of ferrous ion and NADH was discovered and investigated. Kinetic and equilibrium constants for reactions of iron ions - NADH complexation and following redox processes of the complex decomposition were determined from spectrophotometric and electrochemical experiments.;The discovery of significance of iron-NADH complexation for the kinetics of NADH decomposition and oxidation led to an investigation of the influence of solution oxygen and biologically prominent metal ions on NADH degradation. Almost all metal ions under investigation appeared to complex quickly with NADH. Studies of kinetics of simultaneous processes of NADH acid-catalyzed decomposition, complexation, and oxidation in different environments were undertaken. It was observed that Fe2+ and Fe3+ were the only solution ions which caused a decrease in NADH decomposition rates, opposite to all other metal ions under investigation. The presence of metal ions accelerated NADH acid-catalyzed decomposition to a greater extent than NADH oxidation. Contrary to most literature sources, it was determined that the second, and not the first step of decomposition is rate-limiting. (Abstract shortened by UMI.);A 2-channel sensing device capable of simultaneous detection of superoxide radical and hydrogen peroxide in the concentration range 10--7 to 10--4 M was developed. A glassy carbon microelectrode covered by polypyrrole/horseradish peroxidase membrane was employed as a H 2O2 sensor. Another glassy carbon microelectrode covered by a composite membrane of an inside layer of polypyrrole/horseradish peroxidase and an outside layer of superoxide dismutase was employed electrode for superoxide detection. Both sensors along with Pt counter and tungsten oxide reference electrodes were contained in one 6 mm diameter TeflonRTM cylinder. Superoxide sensor behavior as a function of membrane deposition conditions and coating time is reported. Sensors' mutual influence, selectivity, response times, linearity, stability and sensitivity for hydrogen peroxide and superoxide are presented. A mathematical model of sensors' response is proposed, with model calculation corresponding to experiment within 10%.