title: Retention of enzyme activity with a boron-doped diamond electrode in the electro-oxidative nitration of lysozyme.
abstract: In this paper we report the successful use of a non-metallic electrode material, boron-doped diamond (BDD), for the anodic electro-oxidative modification of hen egg white lysozyme (HEWL). Platinum electrodes can give rise to loss of activity of HEWL in electrosynthetic studies, whereas activity is retained on boron-doped diamond which is proposed as an effective substitute material for this purpose. We also compare literature methods of electrode pre-treatment to determine the most effective in electrosynthesis. Our findings show a decrease in total nitroprotein yield with decreasing nitrite concentration and an increase with increasing solution pH, confirming that, at a BDD electrode, the controlling factor remains the concentration of tyrosine phenolate anion. Purification of mono- and bis-nitrated HEWL and assay of enzymic activity showed better retention of activity at BDD electrode surfaces when compared to platinum. The products from electro-oxidation of HEWL at BDD were confirmed by electrospray ionization Fourier transform ion cyclotron resonance (ESI-FT-ICR) mass spectrometry, which revealed unique mass increases of +45 and +90 Da for the mono- and bis-nitrated lysozyme, respectively, corresponding to nitration at tyrosine residues. The nitration sites were confirmed as Tyr23 and Tyr20.
authors.affiliation: Department of Physical Chemistry and Institute of Electrochemistry, University of Alicante, Alicante 03080, Spain.
authors.forename: Jesús
authors.initials: J
authors.lastname: Iniesta
authors.forename: María Deseada
authors.initials: MD
authors.lastname: Esclapez-Vicente
authors.forename: John
authors.initials: J
authors.lastname: Heptinstall
authors.forename: David J
authors.initials: DJ
authors.lastname: Walton
authors.forename: Ian R
authors.initials: IR
authors.lastname: Peterson
authors.forename: Victor A
authors.initials: VA
authors.lastname: Mikhailov
authors.forename: Helen J
authors.initials: HJ
authors.lastname: Cooper