HPLC/CHEMILUMINESCENCE OF OPIOID PEPTIDES

阿片肽的 HPLC/化学发光

• 批准号: 3210410
• 负责人: JOHN F STOBAUGH
• 金额: $ 9.41万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3210410
• 关键词: enkephalins; fluorescent dye /probe; high performance liquid chromatography; laboratory rat; luminescence; neuropeptides

The opioid peptides are biologically important products resulting from the biochemical processing of three precursor proteins: preproopiomelanocortin, preproenkephalin A and preproenkephalin B. The primary objective of this research is the development of direct chemical assay methodology which has the required selectivity and sensitivity to unambiguously quantify opioid peptides, especially, Leu-enkephanlin and Met-enkephalin, in biologically relevant tissues. This overall objective is being accomplished via the unique combination of fluorogenic derivatization, multidimensional HPLC and chemiluminescence detection. Specifically, the primary amine groups of the peptides are reacted with the novel NDA reagent (naphthalene-2,3- dialdehyde plus cyanide) to form stable and intensely fluorescent N-substituted 1-cyanobenz f isoindole (CBI) derivatives. Next, multidimensional chromatography is employed to achieve the desired selectivity. The CBI derivatives are initially fractionated on an HPLC column which retains the analytes by one mechanism (eg., reversed phase) and then switched automatically to a second column with a completely different mechanism (eg., ion-pair). Finally, the resolved analytes are detected via chemiluminescence of the CBI moiety, generated by an aryloxalate/H2O2 post-column reaction detector. The following studies will be carried out to optimize the approach: 1) the rate and yield of the NDA reaction will be studied using several peptides including those with multiple reaction sites, 2) the luminescent properties of the CBI peptides wil be characterized paying particular attention to the products of multiple derivatization, 3) the systematic characterization of the retention of CBI peptides on HPLC columns with different retention mechanisms will be carried out, 4) the characterization of the post-column chemiluminescent process will include: reactor design, flow-cell design, use of novel aryloxalate esters and reaction conditions. The results obtained will permit the unification of these various components into a system that will permit the direct chemical determination of opioid peptides with excellent selectivity and sensitivity (limits of detection of 1-10 femtomole on-column). The resulting methodology will be applicable to the quantification of endogenous or exogenously administered opioid peptides that could be easily transferred to other laboratories engaged in this area of peptide research without the addition of highly specialized equipment.

阿片肽是生物学上重要的产物， 来自三种前体蛋白的生化加工： 前阿黑皮素原、前脑啡肽原A和前脑啡肽原 B。 本研究的主要目的是开发 直接化学分析方法，具有所需的 明确定量阿片样物质的选择性和灵敏度 肽，特别是亮氨酸脑啡肽和蛋氨酸脑啡肽， 生物相关组织。 这一总体目标正在 通过独特的荧光组合完成， 衍生化、多维HPLC和化学发光 侦测 具体地，肽的伯胺基团 与新型NDA试剂（萘-2，3- 二醛加氰化物），形成稳定的强荧光 N-取代的1-氰基苯并[f]异吲哚（CBI）衍生物。 接下来， 多维色谱法是用来实现 所需的选择性。 CBI衍生物最初被分馏 在通过一种机制保留分析物的HPLC色谱柱上 (eg.,然后自动切换到第二个 具有完全不同机制的柱（例如，离子对）。 最后，通过化学发光检测解析的分析物 CBI部分，由芳草酸酯/H2 O2柱后生成 反应检测器 将开展以下研究， 优化方法：1）NDA反应的速率和产率 将使用几种肽进行研究，包括那些 多个反应位点，2）CBI的发光性质 肽的特征在于特别注意 产品的多重衍生化，3）系统 CBI肽在HPLC上保留的表征 将实施具有不同保持机制的柱， 4)柱后荧光的表征 过程将包括：反应器设计，流动池设计，使用新的 芳草酸酯和反应条件。 获得的结果 将允许这些不同的组件统一成一个 该系统将允许直接化学测定 阿片肽具有优异的选择性和灵敏度（限制 1-10飞摩尔柱上检测）。 所得 方法将适用于量化 内源性或外源性给予的阿片肽， 可以很容易地转移到其他实验室从事这一工作， 肽研究领域没有增加高度专业化的 设备.