title: Lysozyme Photochemistry as a Function of Temperature. The Protective Effect of Nanoparticles on Lysozyme Photostability.
abstract: The presence of aromatic residues and their close spatial proximity to disulphide bridges makes hen egg white lysozyme labile to UV excitation. UVB induced photo-oxidation of tryptophan and tyrosine residues leads to photochemical products, such as, kynurenine, N-formylkynurenine and dityrosine and to the disruption of disulphide bridges in proteins. We here report that lysozyme UV induced photochemistry is modulated by temperature, excitation power, illumination time, excitation wavelength and by the presence of plasmonic quencher surfaces, such as gold, and by the presence of natural fluorescence quenchers, such as hyaluronic acid and oleic acid. We show evidence that the photo-oxidation effects triggered by 295 nm at 20°C are reversible and non-reversible at 10°C, 25°C and 30°C. This paper provides evidence that the 295 nm damage threshold of lysozyme lies between 0.1 μW and 0.3 μW. Protein conformational changes induced by temperature and UV light have been detected upon monitoring changes in the fluorescence emission spectra of lysozyme tryptophan residues and SYPRO® Orange. Lysozyme has been conjugated onto gold nanoparticles, coated with hyaluronic acid and oleic acid (HAOA). Steady state and time resolved fluorescence studies of free and conjugated lysozyme onto HAOA gold nanoparticles reveals that the presence of the polymer decreased the rate of the observed photochemical reactions and induced a preference for short fluorescence decay lifetimes. Size and surface charge of the HAOA gold nanoparticles have been determined by dynamic light scattering and zeta potential measurements. TEM analysis of the particles confirms the presence of a gold core surrounded by a HAOA matrix. We conclude that HAOA gold nanoparticles may efficiently protect lysozyme from the photochemical effects of UVB light and this nanocarrier could be potentially applied to other proteins with clinical relevance. In addition, this study confirms that the temperature plays a critical role in the photochemical pathways a protein enters upon UV excitation.
authors.affiliation: Research Center for Biosciences & Health Technologies, Universidade Lusófona, Lisboa, 1749-024, Portugal.|Department of Biomedical Sciences, Faculty of Pharmacy, University of Alcalá, 28871 Alcalá de Henares, Spain.
authors.forename: Catarina
authors.initials: C
authors.lastname: Oliveira Silva
authors.affiliation: Medical Photonics Lab, Department of Health Science and Technology, Faculty of Medicine, Aalborg University, Fredrik Bajers vej 7, DK-9220, Aalborg, Denmark.
authors.forename: Steffen B
authors.initials: SB
authors.lastname: Petersen
authors.affiliation: Research Center for Biosciences & Health Technologies, Universidade Lusófona, Lisboa, 1749-024, Portugal.|IBEB, Biophysics and Biomedical Engineering, Faculty of Sciences, University of Lisbon, 1749-016, Lisbon, Portugal.
authors.forename: Catarina
authors.initials: C
authors.lastname: Pinto Reis
authors.affiliation: Research Center for Biosciences & Health Technologies, Universidade Lusófona, Lisboa, 1749-024, Portugal.
authors.forename: Patrícia
authors.initials: P
authors.lastname: Rijo
authors.affiliation: Department of Biomedical Sciences, Faculty of Pharmacy, University of Alcalá, 28871 Alcalá de Henares, Spain.
authors.forename: Jesús
authors.initials: J
authors.lastname: Molpeceres
authors.affiliation: Department of Ophthalmology, Aalborg University, Hobrovej 18-22, 9000 Aalborg, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Vorum
authors.affiliation: Department of Clinical Medicine, Aalborg University, Søndre Skovvej 15, 9000 Aalborg, Denmark.
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Neves-Petersen
mesh_terms: D000818:Animals; D002642:Chick Embryo; D004527:Egg Proteins; D005453:Fluorescence; D006046:Gold / Q000737:chemistry*; D006820:Hyaluronic Acid / Q000737:chemistry; D053768:Metal Nanoparticles / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry*; D019301:Oleic Acid / Q000737:chemistry; D010084:Oxidation-Reduction; D010777:Photochemistry; D013696:Temperature; D014466:Ultraviolet Rays
chemical_list: D004527:Egg Proteins; D019301:Oleic Acid; D006046:Gold; D006820:Hyaluronic Acid; C421864:hen egg lysozyme; D009113:Muramidase