Product analysis of caffeic acid oxidation by on-line electrochemistry/electrospray ionization mass spectrometry

AbstractOn-line electrochemistry/electrospray ionization mass spectrometry (EC/ESI-MS) was developed using a microflow electrolytic cell. This technique was applied to electrochemical oxidation of caffeic acid (CAF) which is known to be a highly antioxidative agent. Effects of electrolytic potentials on ion intensities of product ions and on electrolytic currents were examined at different pHs. Dimer products were detected at electrolytic potentials of E = 0.7 V (vs. Ag/AgCl) and trimer products at 1.0 V at pH 9. Dimer products were distinguished from hydrogen-bonded complexes by MS/MS experiments. Hydrogen/deuterium exchange experiments determined the number of hydroxyl and carboxyl groups in the Dimers formed by electrolysis. The mechanism of oxidative polymerization of CAF is discussed with speculation as to the structure of the dimer product

Electrochemical characterization of a unique, "neutral" laccase from Flammulina velutipes

The flac1 gene consisted of 1488 bases encodes a novel laccase (Flac1) from Flammulina velutipes. The deduced amino acid sequence of Flac1 with 496 amino acids shows 58-64% homologies with other fungal laccases. The recombinant Flac1 (rFlac1) was heterologously expressed in Pichia pastoris, with sugars of approximately 4 kDa attached on the protein molecule, which has the calculated molecular mass of 53,532 Da. rFlac1 was shown to be a multi-copper oxidase from spectroscopies. The optimum pHs of rFlac1 for oxidations of 2,2\u27-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), p-phenylenediamine, and o-aminophenol, were 5.0, 5.0, and 6.0-6.5, respectively, showing higher pH values than those from many other fungal laccases. The slightly acidic or neutral optimum pH that is not strongly dependent on substrates is a unique property of rFlac1. Effective O2 reduction was realized by the direct electron transfer of rFlac1 at a highly oriented pyrolytic graphite electrode modified with fine carbon particles (Ketjen Black) in O2-saturated solution. The pHs showing the maximum ΔE°\u27 [= E°\u27(enzyme) - E°\u27(substrate)] coincided well with the optimum pHs shown by rFlac1 under steady-state conditions. The present electrochemical results of rFlac1 indicate that ΔE°\u27 is one of the primary factors to determine the activity of multi-copper oxidases. © 2012 The Society for Biotechnology, Japa

Determination of the Electrostatic Potential of Oil-in-Water Emulsion Droplets by Combined Use of Two Membrane Potential-Sensitive Dyes

The fluorescence behaviors of potential-sensitive dyes including anionic DiBAC4(3) (denoted by dye A), DiSBAC2(3) (dye B), and zwitterionic di-4-ANEPPS (dye C) were studied in oil-in-water (O/W) emulsions. In this study, the equilibrium Galvani potential difference (ΔOWφeq) of the O/W-emulsion droplets was controlled by changing the ratio of the concentrations of electrolytes added to the O (=1,2-dichloroethane) and W phases. When using an adequate combination of the dyes, i.e., B and C, we could observe that the ratio of their fluorescence peak intensities was changed from 1.08 to 1.38, depending on the change of (ΔOWφeq from 26 to 73 mV. It is desirable to apply this method to study the potential-dependent ion or electron-transfer reactions occurring at vesicles or liposomes, and also to biomembranes

Determination of the Electrostatic Potential of Oil-in-Water Emulsion Droplets by Combined Use of Two Membrane Potential-Sensitive Dyes

The fluorescence behaviors of potential-sensitive dyes including anionic DiBAC(4)(3) (denoted by dye A), DiSBAC(2)(3) (dye B), and zwitterionic di-4-ANEPPS (dye C) were studied in oil-in-water (O/W) emulsions. In this study, the equilibrium Galvani potential difference (ΔOWφeq) of the O/W-emulsion droplets was controlled by changing the ratio of the concentrations of electrolytes added to the O (=1,2-dichloroethane) and W phases. When using an adequate combination of the dyes, i.e., B and C, we could observe that the ratio of their fluorescence peak intensities was changed from 1.08 to 1.38, depending on the change of (ΔOWφeq from 26 to 73 mV. It is desirable to apply this method to study the potential dependent ion or electron-transfer reactions occurring at vesicles or liposomes, and also to biomembranes