Lysozyme bionanoparticles formed by blue-light sensitized crosslinking: mechanistic insights

VERA, CECILIA; ABATEDAGA, INÉS; REY, VALENTINA; MORAN VIEYRA, F. EDUARDO; BORSARELLI, CLAUDIO DARÍO

Viña del Mar

Encuentro; XIV Encuentro Latinoamericano de Fotoquímica y Fotobiología (ELAFOT); 2019

Local Organizing Committee (Pontificia Universidad Católica de Chile, Universidad de Chile, Universidad Andrés Bello, Universidad de Santiago de Chile)

Photo-induced protein modification can occur via multiple pathways, involving free radical and reactive oxygen species inducing redox reactions mainly of Trp, Tyr, Cys, Met and Hys residues.1In this work, we studied the photosensitized modification of lysozyme (LZ) by blue-light excitation of the ruthenium II complex Ru(bpy)32+ in the presence of a molar excess of S2O82- as a sacrificial electron acceptor.2 Photolysis was performed in N2- or air-saturated buffered solution (pH 7.4) up to 20 min by irradiation with a Royal blue LED (462 ± 14 nm). The reaction was monitored by absorption and fluorescence spectroscopies, laser-flash photolysis, SDS-PAGE and dynamic light scattering analysis, mass spectrometry and also by transmission electronic microscopy.Steady-state spectroscopic changes and MS analysis indicated the efficient formation of protein degradation products, such as di-tyrosine, kynurenine, and quinone derivatives of both residues. The formation of di-tyrosine was favored in N2-saturated solutions. LFP experiments in air-saturated solutions confirmed the efficient quenching of the triplet state of 3Ru(bpy)32+ by S2O82- to form Ru(bpy)33+ and SO4, a strong oxidizing and hydrogen atom abstracting agent.1 In presence of LZ, the changes of the transient absorbance indicated the recovery of the sensitizer due to the reduction of Ru(bpy)33+ and the disappearance of SO4 to SO4H. The photoreaction generates the progressive formation of covalent protein oligomers (e.g. dimer, trimer, ?n-mer) as detected by SDS-PAGE and dynamic light scattering (DLS), determining a size particle increase from approximately 6 to 14 nm. TEM images confirmed the presence of larger globular aggregates after photolysis treatment. Finally, the oligomeric species of LZ showed slight UVA-photosensitizing capability, with singlet oxygen quantum yield of 2%, as determined by the O2-uptake with triptophan and Trp-uptake induced photo-oxidation experiments. In summary, blue-light mediated photosensitization of the Ru(bpy)32+/S2O82- mixture produces soluble oligomeric bionanoparticles of lysozyme that can be used as medical drug and/or dye photosensitizer vehicle for biomedical applications. (1) Pattison, D. I.; Rahmanto, A. S.; Davies, M. J. Photo-Oxidation of Proteins. Photochemical & Photobiological Sciences. The Royal Society of Chemistry 2012, p 38. https://doi.org/10.1039/c1pp05164d.(2) Fancy, D. a; Denison, C.; Kim, K.; Xie, Y.; Holdeman, T.; Amini, F.; Kodadek, T. Scope, Limitations and Mechanistic Aspects of the Photo-Induced Cross- Linking of Proteins by Water-Soluble Metal Complexes. Chem Biol 2000, 7 (9), 697-708.