Photosensitized oxidation of BSA by novel ruthenium polypyridyl complexes with cyano substituents

Abstract

Ruthenium (II) polypyridyl complexes (RPC) display excellent properties as both energyor charge-transfer photosensitizers, which can be modulated with appropriate changes in ligand structure. The well recognized luminescence properties of their metal-to-ligand chargetransfer excited triplet states (3 MLCT*) have led to their applications as suitable photoprobes for sensing and/or modification of biomolecules, such as DNA and proteins [1]. Proteins are the most abundant macromolecules in biological environments. In particular, serum albumins are present in submimolar concentration in blood plasma, where their main function is the transport of fatty acids, among others. Recently, we have explored the photosensitized oxidation of both human (HSA) and bovine (BSA) serum albumins by using absorption and emission spectroscopies [2]. In this work, we explored some aspects of the photosensitized oxidation of BSA by a series of novel mononuclear ruthenium complexes: [Ru(bpy)2(Mebpy-CN)](PF6)2 (1), [Ru(bpy)(Mebpy-CN)2](PF6)2 (2), [Ru(Mebpy-CN)3](PF6)2 (3), with (bpy = 2,2’-bipyridine, Mebpy-CN = 4-methyl-2,2c-bipyridine-4c-carbonitrile) in air-saturated phosphate buffer solutions at pH 7.4 and 25 °C. In spite of the low value of the isoelectric point for BSA (pI = 4.8), all the cationic RPC do not bind to BSA, as demonstrated by the lack of changes in either the emission spectra of the complex or of the protein, in solutions containing up to 30 PM RPC and 200 PM BSA. Under these conditions, the emission anisotropy of the complexes remained unchanged and | 0, as can be expected for a free rotor in fluid media. However, continuous irradiation with blue light (460 nm) of air-saturated aqueous solutions of 30 PM RPC and 120 PM BSA produces only a progressive hyperchromic effect around 300-400 nm, indicating that photolysis modifies BSA but not RPC. The differential absorbance spectra indicated the formation of a new band with maximum at 320 nm. These changes were paralleled with the decrease of almost 50-60% of the fluorescence intensity of BSA, observed by excitation at 295 nm, and with the increase of intensity of a new emission band at 410 nm by excitation at 320 nm. Laser-flash photolysis experiments indicated that the 3 MLCT* of all RPC were efficiently quenched by molecular oxygen (kq O2 = 2-3u109 M-1s-1), and that singlet molecular oxygen (1 O2) was produced with quantum yields higher than that of [Ru(bpy)3](PF6)2, as determined by time-resolved NIR phosphorescence detection in aerated acetonitrile. The above results indicate that the RPC 1-3 are not associated to BSA, but their excited states are capable of producing 1 O2 in the bulk solution, which diffuses to the protein molecule to oxidize Trp residues (among other electron-rich aminoacids, e.g. Cys, His, etc), leading to the formation of secondary oxidation products N-formylkynurenine and HPI (3ahydroxypyrolidinoindole) that give rise to the absorbance at 320 nm and the emission around 410 nm. Therefore, complexes 1-3 are suitable for the photo-oxidation of proteins and subsequently can be easily removed from the modified protein solution by ultrafiltration, dialysis, or ion-exchange chromatography.