Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal.
Arch Biochem Biophys. 2013 Feb 1;530(1):23-31. doi: 10.1016/j.abb.2012.12.010. Epub 2012 Dec 13.
The study of protein damage by oxidative processes and its influence on protein activity is central to understanding the deleterious effects of oxidative stress on biological systems. This paper will focus on the study of enzyme inactivation by oxidative modifications, utilizing α-amylase from Bacillus species. (BAA) as a model protein. Oxidative stress was induced using metal catalyzed oxidation (MCO). The enzymatic activity of BAA was correlated with the oxidative damage induced to the protein. Off-line nano-HPLC-MALDI-TOF/TOF-MS was used to characterize the oxidative modifications occurring to the protein. Additionally, semi-quantitative analysis was employed in order to evaluate the significance of the various oxidative modifications. BAA oxidation was found to be deleterious to its enzymatic activity. A total of 10 amino acid residues were found to have an oxidation degree above 50%, out of which eight were methionine and tryptophan. Residues in the proximity of key structural elements were found to be particularly susceptible to oxidation. The oxidative process was found to be governed by the nature of the amino acid residues side chain and, to a lesser extent, their location within the three dimensional structure of the protein.
蛋白质在氧化过程中受到损伤并对其活性产生影响，这是理解氧化应激对生物系统的有害影响的核心问题。本文将重点研究氧化修饰对酶失活的影响，以芽孢杆菌来源的α-淀粉酶（BAA）作为模型蛋白。利用金属催化氧化（MCO）诱导氧化应激。BAA 的酶活性与蛋白质诱导的氧化损伤相关联。采用离线纳升高效液相色谱-基质辅助激光解吸/飞行时间串联质谱（nano-HPLC-MALDI-TOF/TOF-MS）对蛋白质发生的氧化修饰进行了表征。此外，还进行了半定量分析，以评估各种氧化修饰的意义。BAA 的氧化对其酶活性具有危害性。发现有 10 个氨基酸残基的氧化程度超过 50%，其中 8 个是蛋氨酸和色氨酸。靠近关键结构元素的残基特别容易受到氧化。氧化过程受氨基酸残基侧链的性质控制，在较小程度上受其在蛋白质三维结构中的位置控制。
