Retention of enzyme activity with a boron-doped diamond electrode in the electro-oxidative nitration of lysozyme

• Published in: Enzyme and microbial technology 2010-05, Vol.46 (6), p.472-478
• Main Authors: Iniesta, Jesús, Esclapez-Vicente, María Deseada, Heptinstall, John, Walton, David J., Peterson, Ian R., Mikhailov, Victor A., Cooper, Helen J.
• Format: Article
• Language: English
• Subjects: Albumen; BDD; Biological and medical sciences; Biotechnology; Electro-oxidative nitration; Fundamental and applied biological sciences. Psychology; Infrared multiphoton dissociation; Lysozyme; Nitrotyrosine; Tandem mass spectrometry
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2010.02.002

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 +90Da for the mono- and bis-nitrated lysozyme, respectively, corresponding to nitration at tyrosine residues. The nitration sites were confirmed as Tyr23 and Tyr20.