Photosensitized oxidative crosslinking of bovine serum albumin and the impact on its esterase-like activity

Abstract

A photosensitized oxidative crosslinking of proteins (POCP) reaction was applied in air-saturated phosphate buffer solutions of bovine serum albumin (BSA) to obtain soluble protein nanoparticles of approximately 100 nm in diameter. A royal blue LED was used as the excitation source for the photosensitizer molecule ruthenium (II) tris(2,2′-bipyridyl) dication (Ru(bpy)₃²⁺), in the presence of the electron acceptor persulfate anion (S₂O₈²⁻). The redox quenching products prompted the formation of side-chain tyrosyl radicals, which served as intermediaries in the covalent attachment between proteins, leading to the formation of dityrosine (Tyr₂) links. However, the dissolved oxygen competes efficiently with S₂O₈²⁻ to quench the excited photosensitizer, thereby generating singlet molecular oxygen (¹O₂), which reacts with electron-rich protein residues, which in turn induce an additional oxidative pattern of BSA. Consequently, under air-saturated conditions, the POCP gives rise to a series of oxygen-dependent and -independent reactions, resulting in the protein crosslinking with oxidative modifications. The esterase-like activity efficacy of BSA oxidized solely by ¹O₂ and after the formation of oligomeric protein nanoparticles by POCP was reduced by 51 % and 73 %, respectively, as compared with that of the native BSA. The combination of the oxidative degradation of key residues in the active sites and steric impediment due to protein oligomerization was found to be associated with this result.