A Kinetic Study of the Crosslinking of Lysozyme by Photoinduced Generation of Side-Chain Tyrosyl Radicals

DORADO, LUIS ANDRES; REY, VALENTINA; BORSARELLI, CLAUDIO D.

Mendoza

Congreso; Inter-American Photochemical Society; 2011

Protein oxidation result in severe losses of biological functionality due to changes on the structure and/or conformation of the native protein. Depending on the type of oxidative agent and oxidation degree, the damage of the protein can result in peptide bond breakdown, aminoacid residue modification, crosslinking, etc. Lysozyme (LYZ) or muramidase, a 14.4 kD antimicrobial protein, is very abundant in a number of secretions, such as tears, saliva, human milk, mucus, and in egg white. LYZ has been used as preservative in foods. In the present work, we have studied the formation of covalent oligomeric species of LYZ by  blue-light photosensitization of the metal coordination complex ruthenium (II) tris-bipyridine Ru(bpy)32+ in the presence of ammonium persulfate (APS). Several spectroscopic techniques (stationary and dynamic UV/Vis absorption and fluorescence) together with the analysis of SDS-PAGE were used to characterize the photocrosslinked products of LYZ. The formation of the covalent oligomeric species (dimer, trimer, …n-mer) was proportional with the irradiation time, figure 1. The structural changes were parallel with UV absorption increases at 320 nm, quenching of the intrinsic protein fluorescence at 340 nm together with the increment of fluorescence at 405 nm, figure 2. All this features are indicative of the formation of di-tyrosine (di-Tyr). Laser-flash photolysis experiments demonstrated the formation of tyrosyl radical by electron-transfer reaction of the oxidized Ru(bpy)33+ with Tyr. Finally, the antimicrobial activity of these species was compared with that of native LYZ.