Type I photosensitization of Ubiquitin leads to fluorescent adduct formation, cleavage and dimerization

REID, LARA O.; PETROSELLI GABRIELA; ERRA BALSELLS ROSA; LHIAUBET-VALLET, VIRGINIE; MIRANDA, MIGUEL A; THOMAS ANDRÉS H.; DANTOLA M LAURA

Viña del mar

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

Universidad de Santiago de Chile

Different compounds are able to induce photosensitivity as a result from exposure to certain molecules and light; these includes phototoxic and photoallergic reactions. The photoallergy normally involves a covalent binding between proteins and photosensitizer agents leading to the formation of a complete photoantigen, which may trigger a hypersensitivity reaction due to a cell-mediated immune response.Pterins belong to a family of heterocyclic compounds present in a wide range of living systems and play different roles ranging from pigments to enzymatic cofactors for numerous redox and one-carbon transfer reactions. In pathological conditions, such as vitiligo, a skin disorder characterized by the acquired loss of constitutional pigmentation, pterins accumulate in the affected tissues at concentrations which are significantly higher than those reported for healthy cells.1 Under UV-radiation, this compounds are able to photosensitize damage in proteins, DNA and their components by Type I (electron ?transfer) and Type II (singlet oxygen) mechanisms.1,2 Recently, it has been reported that, in the absence of molecular oxygen, pterin (Ptr) the parent and unsubstituted compound of oxidized pterins, is able to react with thymine (Thy) yielding an adduct Ptr-Thy in which the photosensitizer is covalently linked to the Thy moiety.4Therefore, given the biological and medical relevance of the photosensitizing properties of pterins, the aim of this work is to study if Ptr is able to generate photoadducts with proteins and establish its photoallergic potencial. For this study, aqueous acidic solutions of Ubiquitin (Ub) and Ptr were irradiated (ex=350 nm) at room temperature and in different atmospheres. Ub was used as a model protein given that is a small (8.5 kDa) regulatory protein, which has only one Tyrosine (Tyr) residue and none Tryptophan residue.The irradiated solutions were analyzed by UV/visible spectrophotometry, HPLC, fluorescence spectroscopy, SDS-PAGE and mass spectrometry (MALDI-TOF and nano LC-ESI-Orbitrap).Under UV-A radiation Ptr is able to form an adduct with Ub, and this reaction is much more efficient in the absence of O2. The spectroscopic analysis reveals that the emission and the excitation spectrum are similar to those corresponding to Ptr, as well as the fluorescence lifetime. On the other hand, as a consequence of the photosensitized process, the protein suffers oligomerization mediated by Tyr dimers, and also a fragmentation can occur, which is dependent of the oxygen concentration in the atmosphere.