Characterization of D-amino acid containing protein from sun-damaged skin of elderly donors.

1)日光损伤皮肤弹性纤维中生物学上罕见的D-β-天冬氨酸蛋白，我们制备了D-β-天冬氨酸蛋白抗体，并检测了其在皮肤中的免疫反应性。该抗体识别暴露在阳光下的皮肤中解体的弹性纤维。蛋白质的Western图谱分析表明，该多肽大小为50 kDa，与含有D-β-Asp蛋白和弹性蛋白的抗体呈免疫反应。这一结果表明，在老化过程中，弹性纤维蛋白中残留天冬氨酸的显著消旋和异构化以及异常蛋白的积累。随后，我们通过基质辅助激光解吸电离飞行时间质谱仪和Mascot在线数据库搜索算法鉴定了一些含有D-β-Asp的蛋白质。我们认为这种抗体可以作为皮肤日照损伤的有用指标。2)弹性蛋白切片对应的模型肽中天冬氨酸残基外消旋的动力学研究我们合成了三个与弹性蛋白片段相对应的含有天冬氨酸的模型肽：1)GVADAAAA，2)REGDPSSS，3)AGADEGVR。我们计算了这三种多肽中天冬氨酸残基外消旋的活化能和天冬氨酸D/L在体温(37℃)下达到接近1.0时所需的时间。天冬氨酸在多肽中的消旋反应为一级反应。2号肽中的天冬氨酸残基最容易消旋，而1号肽中的天冬氨酸残基最不容易消旋。但三种多肽对天冬氨酸的消旋率差异不大。在37℃时，这些多肽中天冬氨酸的D/L比值达到0.999所需的时间为50-100年。这些结果表明，天冬氨酸在老年人的皮肤中发生了外消旋。

1)Biologically uncommon D-beta-aspartic acid-containing protein in elastic fibers of sun damaged skin.We have prepared the antibody against D-beta-Asp containing protein and examined its immunoreactivity in skin. The antibody recognized disintegrated elastic fibers in sun-exposed skin. Western plot analysis of the proteins isolated from sun-damaged skin demonstrated that the polypeptide with 50kDa was immunoreactive with the antibodies for D-beta-Asp containing protein and elastin. This result suggests that UV irradiation siginificant racemization and isomerization of Asp resideus in proteins of elastic fibers and the unusual proteins were accumulated during aging. Subsequnetly, we identified some D-beta-Asp containing proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Mascot online database searching algorithm.We propose that the antibody could be a useful indicator for sun damage of the skin.2) Kinetic Study of racemization of aspartyl residues in model peptides corresponding to sections of elastinWe synthesized three Asp-containing model peptides corresponding to sections of elastin:1) GVADAAAA, 2) REGDPSSS, 3) AGADEGVR. We calculated the activation energy of the racemization of Asp residues in the three peptide and the time required for the Asp D/L ratio to approximate to 1.0 at body temperature(37℃). The racemization of Asp in the peptides progressed on the basis of the first order reaction. The Asp residue in the peptide No2 is the most susceptible to racemization, while the Asp residue in the peptide No1 is the least susceptible. However, there was little difference the racemization rate of Asp among three peptides. At 37℃, the time required for the D/L ratios of Asp in these peptides to 0.999 was calculated to be 50-100 years. These results suggested that the racemization of Asp occurred in skin of elderly humans.