Islam K N, Takahashi M, Higashiyama S, Myint T, Uozumi N
Department of Biochemistry, Osaka University Medical School.
J Biochem. 1995 Nov;118(5):1054-60. doi: 10.1093/jb/118.5.1054.
Bovine ceruloplasmin underwent fragmentation following non-enzymatic glycosylation. Western blot and ELISA analyses indicated that a polyclonal rabbit antiserum to hexitolysine reacted with bovine ceruloplasmin after incubation with 0.1 M glucose. The same fragmentation was seen upon exposure of the protein to a hydrogen peroxide bolus. Both catalase and EDTA blocked peroxide-dependent fragmentation. Incubation with glucose resulted in a time-dependent release of Cu2+. The released Cu2+ appeared to participate in a Fenton-type reaction to produce hydroxyl radicals, which effected the fragmentation. Hydroxyl radical scavengers such as thiourea, mannitol, methionine, and formate inhibited this cleavage. ESR spectral studies also supported participation of hydroxyl radicals. Inhibition by EDTA of the fragmentation induced by an H2O2 bolus also supports a role for copper in a Fenton-type reaction. Taken together these results suggest that reactive oxygen species, such as superoxide anion and H2O2, were formed by the Maillard reaction which led to hydroxyl radicals being produced by a copper-dependent Fenton-type reaction. Both processes are likely to be involved in the fragmentation of ceruloplasmin.
牛铜蓝蛋白在非酶糖基化后发生片段化。蛋白质印迹法和酶联免疫吸附测定分析表明，在与0.1M葡萄糖孵育后，针对己糖赖氨酸的兔多克隆抗血清与牛铜蓝蛋白发生反应。将该蛋白质暴露于过氧化氢脉冲时也观察到相同的片段化现象。过氧化氢酶和乙二胺四乙酸均能阻断依赖过氧化物的片段化。与葡萄糖孵育导致铜离子（Cu2+）随时间释放。释放出的铜离子似乎参与了芬顿型反应以产生羟基自由基，从而导致片段化。羟基自由基清除剂如硫脲、甘露醇、蛋氨酸和甲酸盐可抑制这种裂解。电子自旋共振光谱研究也支持羟基自由基的参与。乙二胺四乙酸对过氧化氢脉冲诱导的片段化的抑制作用也支持铜在芬顿型反应中的作用。综合这些结果表明，美拉德反应形成了超氧阴离子和过氧化氢等活性氧，进而通过依赖铜的芬顿型反应产生羟基自由基。这两个过程可能都参与了铜蓝蛋白的片段化。
