Oxidation of bovine serum albumin: identification of oxidation products and structural modifications.
Albumin is an important plasma antioxidant protein, contributing to protecting mechanisms of cellular and regulatory long-lived proteins. The metal-catalyzed oxidation (MCO) of proteins plays an important role during oxidative stress. In this study, we examine the oxidative modification of albumin using an MCO in vitro system. Mass spectrometry, combined with off-line nano-liquid chromatography, was used to identify modifications in amino acid residues. We have found 106 different residues oxidatively damaged, being the main oxidized residues lysines, cysteines, arginines, prolines, histidines and tyrosines. Besides protein hydroxyl derivatives and oxygen additions, we detected other modifications such as deamidations, carbamylations and specific amino acid oxidative modifications. The oxidative damage preferentially affects particular subdomains of the protein at different time-points. Results suggest the oxidative damage occurs first in exposed regions near cysteine disulfide bridges with residues like methionine, tryptophan, lysine, arginine, tyrosine and proline appearing as oxidatively modified. The damage extended afterwards with further oxidation of cysteine residues involved in disulfide bridges and other residues like histidine, phenylalanine and aspartic acid. The time-course evaluation also shows the number of oxidized residues does not increase linearly, suggesting that oxidative unfolding of albumin occurs through a step-ladder mechanism.
Guedes
Sofia
S
Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal.
Vitorino
Rui
R
Domingues
Rosário
R
Amado
Francisco
F
Domingues
Pedro
P
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000032:analysis* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
D000596:Amino Acids; D010455:Peptides; D012710:Serum Albumin, Bovine