Nitration of lysozyme by ultrasonic waves; demonstration by immunochemistry and mass spectrometry

Solutions containing hen egg white lysozyme (HEWL) and nitrite were exposed to ultrasonic irradiation in order to study the possible sonochemical modifications. This is the first demonstration of the nitration of tyrosine residues in a protein (lysozyme) by the use of an ultrasonic field alone. Sonochemically nitrated lysozyme was detected using the immunochemical techniques dot blot immunodetection and enzyme-linked immunosorbent assay (ELISA). The sonically oxidised and nitrated protein solutions were analysed by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. Hydroxylated species were found in the absence of nitrite, whereas nitration was the major modification in the presence of nitrating agent, implying a competing mechanism between hydroxyl radicals and nitrite. Circular dichroism (CD) indicated that the ultrasonic experimental conditions chosen in this study had little effect on the tertiary and secondary structures of HEWL. Whilst enzymatic assay showed that the presence of nitrite provided a protective effect on the inactivation of the protein under ultrasonic irradiation, nevertheless partially purified, sonically nitrated lysozyme showed a dramatic decrease in lytic activity.