HPLC and emission spectroscopy were used to investigate UVA photosensitization of methylene blue (MB) or naproxen (NAP) towards bovine serum albumin (BSA). In addition, time resolved singlet oxygen measurements were carried out. The most stable drug : protein adducts stoichiometry of MB–BSA (1 : 1) and NAP–BSA (9 : 1) were verified by means of binding constant determination. UVA photosensitization of MB or NAP on BSAwas studied by monitoring tryptophan (Trp) residue integrity. The sensitized photodegradation of the BSA resulted in different degrees of Trp damage. Thus, protein damage was determined by quantitative measurements of the different Trp (photo)-products. Indeed, many of these Trp derivatives are diagnostic for the photosensitization mechanism and some of them, for the first time in this work, were obtained by UVA photosensitization in proteins. The analysis of quantum yields of the photoproduct distribution allowed to weigh up the type I/II contribution to the UVA photosensitization mechanism. As expected, additional experiments in deuterated solvent resulted in an increase of the photodegradation quantum yields for those species where a singlet oxygen mechanism was involved. The UVA mediated generation of these Trp derivatives is consistent with the occurrence of singlet oxygen formation (almost dominant in MB), and photoionization (significant in NAP) within the protein matrix. Additional experiments at lower NAP concentration, as well as with human serum albumin (which differs for Trp content and, partially, localization), support further the molecular mechanism of photosensitization proposed. The results obtained in the case of this more complex system are in agreement with the free Trp model, even if, in almost all cases, the Trp photoproduct formation quantum yields are lower, due to the higher number of sensitization targets in the proteins.
|Titolo:||Photoinduced protein modifications by methylene blue and naproxen|
|Data di pubblicazione:||2012|
|Citazione:||Photoinduced protein modifications by methylene blue and naproxen / Bracchitta G; Catalfo A; De Guidi G. - In: PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES. - ISSN 1474-905X. - (2012).|
|Appare nelle tipologie:||1.1 Articolo in rivista|