ANALYSIS OF ENDORPHINS BY LC-ELECTROCHEMISTRY

通过 LC 电化学分析内啡肽

• 批准号: 3210447
• 负责人: NORMAN C REYNOLDS
• 金额: $ 8.54万
• 依托单位: MOUNT SINAI SCHOOL OF MEDICINE OF CUNY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-09-01 至 1991-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3210447
• 关键词: analytical chemistry; brain; dynorphins; electrochemistry; electrodes; endorphins; enkephalins; high performance liquid chromatography; laboratory rat; mass spectrometry; peptides; protein sequence; radioimmunoassay

Efforts to localize and quantify endorphins require techniques that are highly sensitive and specific. Current assays including bioassays, radioreceptor, radioimmunoassay (RIA) and high performance liquid chromatography (LC) with UV detection lack either adequate specificity or adequate sensitivity, particularly for the study of the larger endorphins. LC has been used to separate endorphins in biological samples but quantification is performed by RIA for every endorphin in several chromatographic fractions. The purpose of this project is to develop a specific and quantitative method for the assay of endorphins from small brain regions using LC with electrochemical detection (LC-EC). To accomplish this, the sensitivity of EC detection will be improved using a variety of procedures including alternative electrode configurations (conventional glass carbon, graphite composite, and reticulated vitreous carbon), variation in electrode surface preparation, electrode pre-treatment with anodization procedures and lastly chemical derivatization procedures to improve endorphin electroactivity. The LC-EC assay for endorphins will then be evaluated by several analytical techniques including hydrodynamic voltammetry, peak current ratios, RIA and mass spectrometry to corroborate peak purity and peak identity. The effect of amino acid sequence upon the electroactivity of the N-terminal tyrosyl will be evaluated in a variety of dipeptides and longer peptide sequences to examine both steric and inductive effects upon redox activity. Met- and leu-enkephalin, the dynorphins and -endorphins will be extracted from specific brain regions and the quantitative values obtained with the LC-EC assay compared with those obtained by RIA of the samples. This LC-EC assays will be especially useful for those studies which require a detailed analysis of endorphin content.

定位和量化内啡肽的努力需要技术 高度敏感和特异性。 目前的检测方法包括 生物测定，放射受体，放射免疫测定（RIA）和高 缺少UV检测的高效液相色谱法（LC） 足够的特异性或足够的灵敏度，特别是对于 对更大的内啡肽的研究。 LC已用于分离 生物样品中的内啡肽，但进行定量 通过RIA对几个色谱级分中的每种内啡肽进行分析。 该项目的目的是制定一个具体的， 脑内内啡肽的定量测定方法 区域使用LC电化学检测（LC-EC）。 到 实现这一点，将提高EC检测的灵敏度 使用各种程序包括替代电极 配置（传统的玻璃碳，石墨复合材料， 网状玻璃碳）、电极表面的变化 制备，用阳极氧化程序进行电极预处理 最后是化学衍生化程序， 内啡肽电活性 然后将由几个人评估内啡肽的LC-EC测定法。 分析技术，包括流体动力学伏安法、峰 电流比、RIA和质谱法确证峰 纯度和峰同一性。 氨基酸序列对 N-末端酪氨酰的电活性将在 各种二肽和更长的肽序列来检查 对氧化还原活性的空间效应和诱导效应。 满足-和 亮脑啡肽强啡肽和内啡肽将被提取出来 从特定的大脑区域和获得的定量值 用LC-EC法与RIA法比较， 样本 这种LC-EC测定将特别适用于 那些需要详细分析内啡肽的研究 内容