目的:紫外线(UVB)辐射可以通过各种机制损害皮肤屏障的功能。我们假设UVB诱导皮肤最外层成分的光化学改变,被称为角质层(SC),并调节其抗氧化防御机制。过氧化氢酶是在SC中发现的众所周知的抗氧化酶,其中它起清除活性氧的作用。然而,缺乏急性UVB暴露对SC中天然过氧化氢酶活性的详细表征。此外,UVB照射对SC角蛋白和脂质成分的分子动力学和组织的影响尚不清楚。因此,这项工作的目的是表征UVB暴露对过氧化氢酶的结构和抗氧化特性的影响,以及围绕酶的SC基质的分子和整体特性。
方法:使用皮肤覆盖的氧电极通过计时电流法研究了UVB照射对过氧化氢酶功能的影响,其探测SC基质中天然过氧化氢酶的活性。圆二色性用于探索过氧化氢酶二级结构的变化,和凝胶电泳用于检测UVB暴露后酶的片段化。UVB诱导SC分子动力学和SC屏障结构特征的改变,以及它的吸水行为,通过一套互补的技术进行研究,包括自然丰度13C极化转移固态核磁共振,广角X射线衍射,傅里叶变换红外(FTIR)光谱,和动态蒸汽吸附微天平。
结果:研究结果表明,UVB暴露通过使SC基质中的天然过氧化氢酶和冻干过氧化氢酶均失活而损害过氧化氢酶的抗氧化功能。然而,UVB辐射不会改变过氧化氢酶的二级结构,也不会诱导任何可观察到的酶片段,否则可以解释其功能的失活。对SC样品的NMR测量表明,SC脂质末端片段的分子迁移率略有增加,高剂量UVB暴露后,脂质链反式-gauche构象异构体的迁移率降低。同时,NMR数据表明角蛋白丝的多肽骨架的刚性增加,而角蛋白无规卷曲结构域中氨基酸残基的分子迁移率不受UVB照射的影响。FTIR数据显示与脂质键振动相关的吸光度持续下降,相对于主要的蛋白质条带。总的来说,NMR和FTIR数据表明,在UVB暴露后,SC脂质和蛋白质成分的液相和固相的组成发生了很小的变化,与SC组织的水合能力无关。最后,过氧化氢酶的UVB失活预计会提高SC的氧化应激,which,再加上SC分子特征的细微变化,可能会损害整体皮肤健康,并提高发展为皮肤病的可能性。
OBJECTIVE: Ultraviolet B (UVB) radiation can compromise the functionality of the skin barrier through various mechanisms. We hypothesize that UVB induce photochemical alterations in the components of the outermost layer of the skin, known as the stratum corneum (SC), and modulate its antioxidative defense mechanisms. Catalase is a well-known antioxidative enzyme found in the SC where it acts to scavenge reactive oxygen species. However, a detailed characterization of acute UVB exposure on the activity of native catalase in the SC is lacking. Moreover, the effects of UVB irradiation on the molecular dynamics and organization of the SC keratin and lipid components remain unclear. Thus, the aim of this work is to characterize consequences of UVB exposure on the structural and antioxidative properties of catalase, as well as on the molecular and global properties of the SC matrix surrounding the enzyme.
METHODS: The effect of UVB irradiation on the catalase function is investigated by chronoamperometry with a skin covered oxygen electrode, which probes the activity of native catalase in the SC matrix. Circular dichroism is used to explore changes of the catalase secondary structure, and gel electrophoresis is used to detect fragmentation of the enzyme following the UVB exposure. UVB induced alterations of the SC molecular dynamics and structural features of the SC barrier, as well as its water sorption behavior, are investigated by a complementary set of techniques, including natural abundance 13C polarization transfer solid-state NMR, wide-angle X-ray diffraction, Fourier transform infrared (FTIR) spectroscopy, and dynamic vapor sorption microbalance.
RESULTS: The findings show that UVB exposure impairs the antioxidative function of catalase by deactivating both native catalase in the SC matrix and lyophilized catalase. However, UVB radiation does not alter the secondary structure of the catalase nor induce any observable enzyme fragmentation, which otherwise could explain deactivation of its function. NMR measurements on SC samples show a subtle increase in the molecular mobility of the terminal segments of the SC lipids, accompanied by a decrease in the mobility of lipid chain trans-gauche conformers after high doses of UVB exposure. At the same time, the NMR data suggest increased rigidity of the polypeptide backbone of the keratin filaments, while the molecular mobility of amino acid residues in random coil domains of keratin remain unaffected by UVB irradiation. The FTIR data show a consistent decrease in absorbance associated with lipid bond vibrations, relative to the main protein bands. Collectively, the NMR and FTIR data suggest a small modification in the composition of fluid and solid phases of the SC lipid and protein components after UVB exposure, unrelated to the hydration capacity of the SC tissue. To conclude, UVB deactivation of catalase is anticipated to elevate oxidative stress of the SC, which, when coupled with subtle changes in the molecular characteristics of the SC, may compromise the overall skin health and elevate the likelihood of developing skin disorders.