Use of recombinant iron-superoxide dismutase as a marker of nitrative stress.
Superoxide dismutases (SODs; EC 1.15.1.1) are a group of metalloenzymes which are essential to protect cells under aerobic conditions. In biological systems, it has been reported that SODs and other proteins are susceptible to be attacked by peroxynitrite (ONOO(-)) which can be originated from the reaction of nitric oxide with superoxide radical. ONOO(-) is a strong oxidant molecule capable of nitrating peptides and proteins at the phenyl side chain of the tyrosine residues. In the present work, bovine serum albumin (BSA) and recombinant iron-superoxide dismutase from the plant cowpea (Vu_FeSOD) are used as target molecules to estimate ONOO(-) production. The method employs the compound SIN-1, which simultaneously generates *NO and O(2)(-) in aerobic aqueous solutions. First, assay conditions were optimized incubating BSA with different concentrations of SIN-1, and at a later stage, the effect on the tyrosine nitration and catalytic activity of Vu_FeSOD was examined by in-gel activity and spectrophotometric assays. Both BSA and Vu_FeSOD are nitrated in a dose-dependent manner, and, at least in BSA nitration, the reaction seems to be metal catalyzed.
Larrainzar
Estíbaliz
E
Departamento de Ciencias del Medio Natural, Universidad Pública de Navarra, Campus de Arrosadía, E-31006 Pamplona, Navarre, Spain.
Urarte
Estíbaliz
E
Auzmendi
Iñigo
I
Ariz
Idoia
I
Arrese-Igor
Cesar
C
González
Esther M
EM
Moran
Jose F
JF
D000906:Antibodies / Q000494:pharmacology; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D007150:Immunohistochemistry / Q000379:methods; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009574:Nitro Compounds / Q000032:analysis / Q000378:metabolism; D015538:Nitrosation; D018384:Oxidative Stress; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013482:Superoxide Dismutase / Q000032:analysis* / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D015415:Biomarkers; D009574:Nitro Compounds; D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine; C002744:3-nitrotyrosine; D014443:Tyrosine; C002385:linsidomine; D008981:Molsidomine; D013482:Superoxide Dismutase