Photochemical and Biological Consequences of the Photocrosslinking of Lysozyme

LUIS A. DORADO, VALENTINA REY, ANA LEDESMA, BEATRIZ LOPEZ DE MISHIMA, CARLA E. GIACOMELLI, CLAUDIO D. BORSARELLI

Salta, Argentina

Congreso; 3rd Latin American Protein Society Meeting; 2010

Photochemical and Biological Consequences of the Photocrosslinking of Lysozyme Luis A. Dorado, Valentina Rey, Ana Ledesma, Beatriz Lopez de Mishima, Carla E. Giacomelli2, Claudio D. Borsarelli   cInstituto de Química del NOA (INQUINOA),Facultad de Agronomía y Agroindustrias, Universidad Nacional de Santiago del Estero, Av. Belgrano (S) 1912. 4200-Sgo del Estero, Argentina. E-mail: luisdorado79@hotmail.com 2 Instituto de Investigaciones en Fisicoquímica de Córdoba (INFIQC), Facultad de Ciencias Químicas, UNC.     Protein oxidation results 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, intra- and/or 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. However, LYZ is unstable in solution in combination with a sensitizer, such as riboflavin, and visible light. In the present work, we have studied the formation of covalent oligomeric species of LYZ by action of protein tyrosyl radicals generated by blue-light photosensitization of the metal coordination complex ruthenium (II) tris-bipyridine (Rubpy) 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 covalent oligomeric species (dimer, trimer, …n-mer) was proportional with the irradiation time. The structural changes were parallel with the quenching of the intrinsic tryptophan (Trp) emission at 340 nm together with the increment of the typical di-tyrosine (di-Tyr) emission at 405 nm. Therefore, the Trp and Tyr residues participate in the photoinduced formation of LYZ oligomeric species. The antimicrobial activity of these species was compared with that of native LYZ.   Acknowledgments: authors gratefully acknowledges to CONICET for financial support. CDB also thanks FONCyT for supporting the PICT2006-01090