Site-specific and random fragmentation of Cu,Zn-superoxide dismutase by glycation reaction. Implication of reactive oxygen species.
Site-specific and random fragmentation of human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) was observed following the glycation reaction (the early stage of the Maillard reaction). The fragmentation proceeded in two steps. In the first step, Cu,Zn-SOD was cleaved at a peptide bond between Pro62 and His63, as judged by amino acid analysis and sequencing of fragment peptides, yielding a large (15 kDa) and a small (5 kDa) fragment. In the second step, random fragmentation occurred. The ESR spectrum of the glycated Cu,Zn-SOD suggested that reactive oxygen species was implicated in the both steps of fragmentation. The same fragmentations were seen upon exposure of the enzyme to an H2O2 bolus. Catalase completely blocked both steps of the fragmentation process, whereas EDTA blocked only the second step. Incubation with glucose resulted in a time-dependent release of Cu2+ from the Cu,Zn-SOD molecule. The released Cu2+ then likely participated in a Fenton's type of reaction to produce hydroxyl radical, which may cause the nonspecific fragmentation. Evidence that EDTA abolished only the second step of fragmentation induced by an H2O2 bolus supports this mechanism. This is the first report that a site-specific fragmentation of a protein is caused by reactive oxygen species formed by the Maillard reaction.
Ookawara
T
T
Department of Biochemistry, Osaka University Medical School, Japan.
Kawamura
N
N
Kitagawa
Y
Y
Taniguchi
N
N
D000595:Amino Acid Sequence; D002412:Cations; D003300:Copper / Q000032:analysis; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005609:Free Radicals; D005947:Glucose / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000737:chemistry; D015416:Maillard Reaction; D008969:Molecular Sequence Data; D010100:Oxygen / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D013481:Superoxides / Q000737:chemistry; D014969:Xanthine Oxidase / Q000737:chemistry
D002412:Cations; D005609:Free Radicals; D007042:Hypoxanthines; D010446:Peptide Fragments; D013481:Superoxides; D019271:Hypoxanthine; D003300:Copper; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D005947:Glucose; D010100:Oxygen