Nicotinamide adenine dinucleotide species in the horseradish peroxidase-oxidase oscillator.

NADH chemistry ancillary to the oscillatory peroxidase-oxidase (PO) reaction has been reexamined. Previously, (NAD)2 has been thought of as a terminal, inert product of the PO reaction. We now show that (NAD)2 is a central reactant in this system. Although we found traces of the dimer after several hours of the PO reaction, no accumulation of the dimer occurred, regardless of the reaction time or the number of oscillations. (NAD)2 can convert horseradish peroxidase (HRP) compound I (CpI) to compound II (CpII) with apparent rate constant (2.7 +/- 0.2) x 105 M-1.s-1 and CpII to HRP at 1 x 105 M-1.s-1. Moreover, a reduction of HRP compound III (CpIII) to CpI by (NAD)2 occurs with a rate constant faster than 5 x 106 M-1.s-1. The (NAD)2 reduction of CpIII provides an alternative to the reduction by NAD radical suggested by Yokota and Yamazaki. HRP catalyzes oxidation of alpha-NADH, not only the beta anomer as previously assumed. Rate constants of alpha- and beta-NADH reactions with CpI are (7.4 +/- 0.4) x 105 M-1.s-1, and (1.7 +/- 0.2) x 105 M-1.s-1, and with CpII are estimated as 5 x 104 M-1.s-1, and 4 x 104 M-1.s-1. Apparent rate constants of reduction of methylene blue (MB) to leuco-methylene blue (MBH) are 3.8 x 104 M-1.s-1 for NADH and 6.4 x 104 M-1.s-1 for NAD dimer, (NAD)2, while reoxidation of MBH proceeds at (2.1 +/- 0.2) x 103 M-1.s-1 All the rates were measured in 0.1 M acetate buffer, pH 5.1.

Principal component analysis of dynamical features in the peroxidase-oxidase reaction

Inherent variance due to oscillations in the peroxidase-oxidase (PO) reaction was studied using principal component analysis (PCA). The substrates were oxygen and reduced nicotinamide adenine dinucleotide (NADH). Horseradish peroxidase (HRP) catalyzed the reaction. The concentration of a cofactor, methylene blue (MB), was varied, and 2,4-dichlorophenol was kept constant. Increase in the NADH influx was used to change the reaction dynamics from periodic to chaotic. The reaction space was abstracted to the most significant, mutually independent, pairs of absorption and kinetic basis vectors (principal components). Typically, two significant principal components were extracted from the periodic time series and three from the chaotic data. The PCA models accounted for 70-97% of experimental variance. The greatest fraction of the total variance was accounted for in experiments exhibiting periodic dynamics and less than 25 nM MB. More MB induced an increased contribution of NADH to the PO oscillator variance, as did increased NADH influx. A simulated absorption time series, computed from a mass-action model of the chemistry, was analyzed by PCA as well. The comparison of simulation with experiment indicates that the chemical model renders the time series for HRP oxidation forms with fidelity, but incompletely represents NADH chemistry and other salient processes underlying the observed dynamics.

Amperometric sensors for simultaneous superoxide and hydrogen peroxide detection.

A two-channel sensor capable of almost instantaneous simultaneous detection of superoxide radical and hydrogen peroxide in the concentration range 10(-)(7)-10(-)(4) M is very important for understanding of a number of rapid kinetics processes. A glassy carbon working microelectrode covered by an electrodeposited polypyrrole/horseradish peroxidase (PPy/HRP) membrane was employed as a H(2)O(2) sensor. Another glassy carbon microelectrode covered by a composite membrane of an inside layer of PPy/HRP and an outside layer of superoxide dismutase was employed as a working electrode for superoxide detection. These two working electrodes with Pt counter and tungsten oxide (WO(3)) reference electrodes were contained in one 6 mm diameter Teflon cylinder. Simultaneous measurements were performed at a potential of -60 mV (vs WO(3) reference, pH 5.1). Additional sensor characterization was performed for pH 5.1-9.0. 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 and discussed. A mathematical model of sensors' responses is proposed, with model calculation corresponding to experiment within 10%.

Complexation of nicotinamide adenine dinucleotide with ferric and ferrous ions.

Motivated by the observed influence of stainless steel and ferric and ferrous ions on the behavior of the peroxidase/oxidase oscillator, the mechanism and kinetics of interaction of 1,4-dihydronicotinamide adenine dinucleotide (NADH) with iron ions in 0.1 M acetic acid/sodium acetate buffer with pH 5.1 and with the solution/stainless steel interface were extensively studied. The character of a possible mutual influence of NADH/acetate buffer solution and Type 316 stainless steel has been investigated. We also suggest the mechanism of stainless steel corrosion inhibition by NADH. It was determined that fast complexation of ferric and ferrous ions with NADH occurred with rate constant kcompl = 4.0 x 10(9) +/- 0.2 x 10(9) M-1 s-1. The composition of the product complex is [Fe-(NADH)2] for both Fe2+ and Fe3+. A previously unreported complex of ferrous ion and NADH was discovered, determined, and separately 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.

Active polarization compensation and goniometer for angularly resolved light scattering measurements.

A method for active polarization compensation for a scanning goniometer is presented. This is part of instrumentation constructed for measurements of light scattering by particulates in a high voltage spark. Individual optical element characterization is made using ellipsometry. Mueller matrix calculations are used to model the optical system. The inverse of the optical system is used to calculate the necessary input polarization state. A polarized source with angularly controlled halfwave and quarterwave retarders is used to introduce the necessary polarization state into the goniometer.