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

9727613 Branchaud最近，在几种蛋白质中发现了不可逆的氧化氨基酸侧链交联。 这种交联可以在轻度氧化生理条件下自发发生。 氧化性氨基酸侧链交联可用于有用的目的，为蛋白质提供结构稳定性（酪氨酸酶）或在酶活性位点提供新的内置氧化还原辅因子（半乳糖氧化酶、乙二醛氧化酶和甲胺脱氢酶）. 氧化性氨基酸侧链交联也是氧化代谢的不期望但不可避免的结果。 例如，邻酪氨酸和二酪氨酸被认为是氧化应激和衰老过程的标志物。 氨基酸侧链的不可逆氧化交联可能比目前认为的更普遍。 使用现有方法分析蛋白质的氨基酸组成时，很容易忽略这种交联。 本研究将发展一种新的、通用的、高灵敏度的荧光检测方法，用于蛋白质中侧链交联氨基酸的荧光检测，该方法利用芘形成的激基缔合物，使侧链交联氨基酸“发光”，并在完整蛋白质的荧光HPLC分析中在正常氨基酸的背景下独特地突出。 分离纯化的HPLC级分的质谱分析将鉴定交联的氨基酸组成和化学结构。 食物的燃烧提供了维持生命所必需的能量。大约1-6%的用于燃烧食物的氧气没有完全消耗，而是产生活性氧（ROS）。 ROS会破坏体内的分子、细胞和器官。 该研究项目将开发一种新的方法来检测蛋白质的氧化损伤。 蛋白质是生物的主要组成部分。 该方法在基础生物化学和应用生物医学研究中有许多可能的应用。

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.