Ookawara T, Kawamura N, Kitagawa Y, Taniguchi N
Department of Biochemistry, Osaka University Medical School, Japan.
J Biol Chem. 1992 Sep 15;267(26):18505-10.
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.
在糖基化反应（美拉德反应的早期阶段）之后，观察到了人铜锌超氧化物歧化酶（Cu,Zn-SOD）的位点特异性和随机片段化。片段化过程分两步进行。第一步，通过对片段肽的氨基酸分析和测序判断，Cu,Zn-SOD在Pro62和His63之间的肽键处被切割，产生一个大片段（15 kDa）和一个小片段（5 kDa）。第二步，发生随机片段化。糖基化的Cu,Zn-SOD的电子顺磁共振光谱表明，活性氧参与了片段化的两个步骤。将该酶暴露于过氧化氢脉冲时也观察到相同的片段化。过氧化氢酶完全阻断了片段化过程的两个步骤，而乙二胺四乙酸（EDTA）仅阻断第二步。与葡萄糖一起孵育导致Cu2+从Cu,Zn-SOD分子中随时间释放。释放出的Cu2+随后可能参与了类芬顿反应以产生羟基自由基，这可能导致非特异性片段化。EDTA仅消除过氧化氢脉冲诱导的片段化第二步的证据支持了这一机制。这是首篇报道美拉德反应形成的活性氧导致蛋白质位点特异性片段化的文章。
