Akagawa Mitsugu, Sasaki Daisuke, Kurota Yayoi, Suyama Kyozo
Division of Bioscience and Biotechnology for Future Bioindustries, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan.
Ann N Y Acad Sci. 2005 Jun;1043:129-34. doi: 10.1196/annals.1333.016.
Recent research has demonstrated that nonenzymatic glycation (the Maillard reaction) lead to the formation of carbonyl groups and advanced glycation end products (AGEs) in proteins. Such oxidative modifications are a major contributing factor to diabetic complications and aging. alpha-Aminoadipic semialdehyde (AAS) and gamma-glutamic semialdehyde (GGS) have been identified as the major carbonyl products in oxidized proteins both in vitro and in vivo. AAS is an oxidative deamination product of lysine residue, while GGS originates from arginine and proline residues. To evaluate oxidative damage to proteins by the Maillard reaction, we developed a method of detecting AAS and GGS by high-performance liquid chromatography (HPLC). The aldehydic residues in proteins were derivatized by reductive amination with NaCNBH3 and p-aminobenzoic acid (ABA), a fluorescence regent. After acid hydrolysis of the ABA-derivatized protein, ABA-AAS and ABA-GGS were measured by fluorometric HPLC. Thus, AAS and GGS could be detected in various proteins such as human plasma protein using the present method. Accumulation of both aldehydic residues was observed in oxidized proteins by reactive oxygen species. Furthermore, AAS and GGS were markedly formed in the incubation of BSA with ascorbic acid. The formation of both aldehydic residues was also observed in the incubation of BSA with 100 mM glucose or 1.0 mM methylglyoxal in the absence and presence of 100 microM Fe3+ for 2 weeks. These results suggest that the Maillard reaction can contribute to the formation of AAS and GGS in vivo.
最近的研究表明，非酶糖基化（美拉德反应）会导致蛋白质中羰基基团和晚期糖基化终产物（AGEs）的形成。这种氧化修饰是糖尿病并发症和衰老的主要促成因素。α-氨基己二酸半醛（AAS）和γ-谷氨酸半醛（GGS）已被确定为体外和体内氧化蛋白质中的主要羰基产物。AAS是赖氨酸残基的氧化脱氨产物，而GGS则源自精氨酸和脯氨酸残基。为了评估美拉德反应对蛋白质的氧化损伤，我们开发了一种通过高效液相色谱（HPLC）检测AAS和GGS的方法。蛋白质中的醛基残基通过用NaCNBH3和荧光试剂对氨基苯甲酸（ABA）进行还原胺化反应进行衍生化。在对ABA衍生化的蛋白质进行酸水解后，通过荧光HPLC测定ABA-AAS和ABA-GGS。因此，使用本方法可以在各种蛋白质（如人血浆蛋白）中检测到AAS和GGS。通过活性氧在氧化蛋白质中观察到了这两种醛基残基的积累。此外，在牛血清白蛋白（BSA）与抗坏血酸的孵育过程中明显形成了AAS和GGS。在100 microM Fe3+存在和不存在的情况下，将BSA与100 mM葡萄糖或1.0 mM甲基乙二醛孵育2周，也观察到了这两种醛基残基的形成。这些结果表明，美拉德反应可能在体内促成AAS和GGS的形成。
