title: Interaction of singlet oxygen with bovine serum albumin and the role of the protein nano-compartmentalization.
abstract: Singlet molecular oxygen ((1)O2) contributes to protein damage triggering biophysical and biochemical changes that can be related with aging and oxidative stress. Serum albumins, such as bovine serum albumin (BSA), are abundant proteins in blood plasma with different biological functions. This paper presents a kinetic and spectroscopic study of the (1)O2-mediated oxidation of BSA using the tris(2,2'-bipyridine)ruthenium(II) cation [Ru(bpy)3](2+) as sensitizer. BSA quenches efficiently (1)O2 with a total (chemical+physical interaction) rate constant kt(BSA)=7.3(±0.4)×10(8)M(-1)s(-1), where the chemical pathway represented 37% of the interaction. This efficient quenching by BSA indicates the participation of several reactive residues. MALDI-TOF MS analysis of intact BSA confirmed that after oxidation by (1)O2, the mass protein increased the equivalent of 13 oxygen atoms. Time-resolved emission spectra analysis of BSA established that Trp residues were oxidized to N'-formylkynurenine, being the solvent-accessible W134 preferentially oxidized by (1)O2 as compared with the buried W213. MS confirmed oxidation of at least two Tyr residues to form dihydroxyphenylalanine, with a global reactivity towards (1)O2 six-times lower than for Trp residues. Despite the lack of MS evidences, kinetic and chemical analysis also suggested that residues other than Trp and Tyr, e.g. Met, must react with (1)O2. Modeling of the 3D-structure of BSA indicated that the oxidation pattern involves a random distribution of (1)O2 into BSA; allowing also the interaction of (1)O2 with buried residues by its diffusion from the bulk solvent through interconnected internal hydrophilic and hydrophobic grooves.
authors.affiliation: Instituto de Bionanotecnología, INBIONATEC-CONICET, Universidad Nacional de Santiago del Estero (UNSE), RN9, Km 1125, G4206XCP Santiago del Estero, Argentina.
authors.forename: Rodrigo E
authors.initials: RE
authors.lastname: Giménez
authors.affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic.
authors.forename: Veronika
authors.initials: V
authors.lastname: Vargová
authors.affiliation: Instituto de Bionanotecnología, INBIONATEC-CONICET, Universidad Nacional de Santiago del Estero (UNSE), RN9, Km 1125, G4206XCP Santiago del Estero, Argentina.
authors.forename: Valentina
authors.initials: V
authors.lastname: Rey
authors.affiliation: Instituto de Bionanotecnología, INBIONATEC-CONICET, Universidad Nacional de Santiago del Estero (UNSE), RN9, Km 1125, G4206XCP Santiago del Estero, Argentina.
authors.forename: M Beatriz Espeche
authors.initials: MB
authors.lastname: Turbay
authors.affiliation: Instituto de Bionanotecnología, INBIONATEC-CONICET, Universidad Nacional de Santiago del Estero (UNSE), RN9, Km 1125, G4206XCP Santiago del Estero, Argentina.
authors.forename: Inés
authors.initials: I
authors.lastname: Abatedaga
authors.affiliation: Instituto de Bionanotecnología, INBIONATEC-CONICET, Universidad Nacional de Santiago del Estero (UNSE), RN9, Km 1125, G4206XCP Santiago del Estero, Argentina.
authors.forename: Faustino E
authors.initials: FE
authors.lastname: Morán Vieyra
authors.affiliation: Instituto de Bionanotecnología, INBIONATEC-CONICET, Universidad Nacional de Santiago del Estero (UNSE), RN9, Km 1125, G4206XCP Santiago del Estero, Argentina.
authors.forename: Verónica I
authors.initials: VI
authors.lastname: Paz Zanini
authors.affiliation: INQUINOA-CONICET, Instituto de Química Física, Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Ayacucho 471, T4000INI San Miguel de Tucumán, Argentina.
authors.forename: Juan H
authors.initials: JH
authors.lastname: Mecchia Ortiz
authors.affiliation: INQUINOA-CONICET, Instituto de Química Física, Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Ayacucho 471, T4000INI San Miguel de Tucumán, Argentina.
authors.forename: Néstor E
authors.initials: NE
authors.lastname: Katz
authors.affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic.
authors.forename: Veronika
authors.initials: V
authors.lastname: Ostatná
authors.affiliation: Instituto de Bionanotecnología, INBIONATEC-CONICET, Universidad Nacional de Santiago del Estero (UNSE), RN9, Km 1125, G4206XCP Santiago del Estero, Argentina. Electronic address: cdborsarelli@gmail.com.
authors.forename: Claudio D
authors.initials: CD
authors.lastname: Borsarelli
mesh_terms: D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives / Q000494:pharmacology; D000375:Aging / Q000378:metabolism* / Q000473:pathology; D056831:Coordination Complexes; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011485:Protein Binding; D012710:Serum Albumin, Bovine / Q000737:chemistry* / Q000378:metabolism; D026082:Singlet Oxygen / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism; D017975:Ruthenium Compounds
chemical_list: D015082:2,2'-Dipyridyl; D056831:Coordination Complexes; D012710:Serum Albumin, Bovine; D026082:Singlet Oxygen; D014364:Tryptophan; C547232:tris(2,2-bipyridine)-ruthenium(II); D017975:Ruthenium Compounds
keywords: Albumin; Photosensitization; Protein oxidation; Reactive oxygen species; Singlet oxygen; Tryptophan fluorescence