SGER: Development of a Rapid and Sensitive Fluorescence- Based Method to Detect and Characterize Irreversible Oxidative Sidechain Crosslinks in Proteins

SGER：开发一种快速、灵敏的荧光方法来检测和表征蛋白质中不可逆氧化侧链交联

• 批准号: 9808017
• 负责人: Bruce Branchaud
• 金额: $ 5万
• 依托单位: University of Oregon Eugene
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-15 至 2000-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9808017&HistoricalAwards=false
• 关键词: SGER; Development; Rapid; Sensitive; Fluorescence

9727613 Branchaud Recently, irreversible oxidative amino acid sidechain crosslinks have been found in several proteins. Such crosslinks can occur spontaneously under mildly oxidative physiological conditions. Oxidative amino acid sidechain crosslinks can serve useful purposes, to provide structural stability to proteins (tyrosinase) or to provide novel built- in redox cofactors at enzyme active sites (galactose oxidase, glyoxal oxidase, and methylamine dehydrogenase). Oxidative amino acid sidechain crosslinks are also an undesired but inevitable consequence of oxidative metabolism. For example, ortho-tyrosine and dityrosine are believed to be markers for oxidative stress and for aging processes. Irreversible oxidative crosslinking of amino acid sidechains may be more widespread than currently believed. Such crosslinks could be easily overlooked using existing methods to analyze for amino acid composition of proteins. This research will develop a novel, general and highly sensitive method for fluorescence detection of sidechain crosslinked amino acids in proteins utilizing excimer formation by pyrene to make the sidechain crosslinked amino acids "light up" and uniquely stand out against a background of normal amino acids in fluorescent HPLC analysis of complete protein digests. Mass spec troscopic analysis of isolated purified HPLC fractions will identify the amino acid compositions and chemical structures of crosslinks. Combustion of food provides the energy necessary to support life. Approximately 1-6% of the oxygen used to burn food is not completely consumed but instead produces reactive oxygen species (ROS). ROS damage molecules, cells and organs in the body. This research project will develop a new method to detect oxidative damage to proteins. Proteins are major building blocks of living things. There are many possible applications of the method both in fundamental biochemical and applied biomedical research.

9727613 Branchaud最近，在几种蛋白质中发现了不可逆的氧化氨基酸SIDECHAIN交联。 这种交联可以在轻度氧化的生理条件下自发发生。 氧化氨基酸Sidechain交联可以实现有用的目的，为蛋白质（酪氨酸酶）提供结构稳定性，或在酶活性位点（半乳糖氧化酶，乙醇氧化酶，乙二醇氧化酶和甲基胺脱氢酶）提供新颖的氧化还原辅助因子内置。 氧化氨基酸Sidechain交联也是氧化代谢的不希望但不可避免的结果。 例如，据信矫正酪氨酸和二苯胺是氧化应激和衰老过程的标记。 氨基酸侧链的不可逆氧化交联可能比目前认为的更为普遍。 使用现有方法来分析蛋白质的氨基酸组成，可以很容易地忽略此类交联。 这项研究将开发一种新型，一般且高度敏感的方法，用于利用pyrene的蛋白质中侧链交联氨基酸的荧光检测，以使Sidechain交联的氨基酸“点亮”，并在荧光HPLC分析蛋白蛋白含量分析中与正常氨基酸的背景相对于正常氨基酸的背景。 分离纯化的HPLC级分的质量规格分析将确定交联的氨基酸组成和化学结构。 食物的燃烧提供了支持生活所需的能量。用来燃烧食物的氧气中约有1-6％的氧不是完全消耗的，而是产生活性氧（ROS）。 ROS损害体内的分子，细胞和器官。 该研究项目将开发一种检测蛋白质氧化损害的新方法。 蛋白质是生物的主要基础。 该方法在基本生化和应用生物医学研究中都有许多可能的应用。