Selective modification at tyrosine residues by the electrochemical, sonoelectrochemical and sonochemical nitration of lysozyme Walton, David J. 1; Heptinstall, John1, Mercer, Sadie1, Peterson, Ian R.1; Matters, Dominic1; Esclapez-Vicente, Mº Deseada2; Iniesta, Jesús2 Cooper, Helen J3 1School of Science and the Environment, Center for Molecular and Biomedical Science, Coventry University, Coventry CV1 5FB, United Kingdom 2Physical Chemistry Department, Alicante University, Alicante 03080, Spain 3 School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom. Examination of the electrochemical, sonoelectrochemical and sonochemical modification of proteins and other bioactive molecules may result in the production of novel proteins, enzymes and other bioactive species, in comparison with traditional methodologies such as protein engineering and the use of chemical reagents. The aim of this is to produce electrosynthetically nitrated proteins and to manipulate the nature of that modification either by the use of ultrasound irradiation during electrolysis or by simple exposure of an aqueous protein solution to an ultrasonic field. We show that the above techniques have various consequences for the labelling of proteins, for their specific immobilisation, use in biosensors, the production of novel modified proteins for pathophysiology of oxidative dysfunction, and the microencapsulation of proteins in polymeric microspheres. Hen egg white lysozyme (HEWL) was treated in pH9 aqueous buffer by electrochemical, sonoelectrochemical and sonochemical reaction in the presence of sodium nitrite. Site specific nitration and the stability of lysozyme during these treatments was evaluated by UV-vis spectroscopy, HPLC, direct mass spectrometry, enzyme linked immunosorbent assay (ELISA) and by determining the enzyme activity before and after nitration. In earlier work we showed the electrochemical nitration of HEWL to be selective for tyrosine 23 initially, followed by bisnitration at tyrosine 20 with no trisnitration at tyrosine 53 [1]. Now in this presentation we assess the simultaneous ultrasonic irradiation of the electronitration of HEWL using a 40 kHz 180 w ultrasonic cleaning bath, resulting in different mono to bis nitration ratios at the same residues compared to those in a silent system. Sonication of aqueous lysozyme solutions with and without sodium nitrite was also performed and to the best of our knowledge we show for the first time the sonochemical nitration of HEWL in the presence of a nitrogen source such nitrite [2].

Keywords: Lysozyme, protein stability, ultrasound, free radicals, nitration.

[1] D.J. Walton, J. Heptinstall, (2000), Prep. Biochem. Biotechnol. 30; 1-14.
[2] D Matters, H J. Cooper, L McDonnell, J Iniesta, J Heptinstall, PJ Derrick, DJ Walton, I Peterson. Submitted to Anal. Biochem.

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1. Research in the group "New Technological Development in Electrochemistry: Sonoelectrochemistry and Bioelectrochemistry" Alicante University
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