LOW TEMPERATURE CHEMISTRY/KINETICS/PHOTOAFFINITY LABELS

低温化学/动力学/光亲和标签

• 批准号: 3286473
• 负责人: MATTHEW S PLATZ
• 金额: $ 6.37万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1989-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3286473
• 关键词: affinity labeling; azides; chemical reaction; chymotrypsin; diazo compound; electron spin resonance spectroscopy; free radicals; freezing; hydrogen transport; photochemistry; quantum chemistry

We intend to covalently modify a-chymotrypsin with various aryl azides and diazocompounds. Crystals of the pure modified proteins will be photolyzed at -196 degrees. We will use ESR spectroscopy to directly detect the resultant enzyme bound nitrenes and carbenes. We will use ESR to measure the kinetics of reaction of the nitrenes and carbenes with the enzyme. We expect the reaction will involve hydrogen atom transfer by quantum mechanical tunneling to give a radical-pair. The kinetic barrier will be fit to an Eckart barrier to obtain the barrier width-the distance travelled by the tunneling hydrogen from an amino acid residue to the carbene or nitrene. We will detect the product radical pair by ESR. The spectrum of the radical pair will give a second measure of the separation of the pair. The nuclear hyperfine interaction present in the pair will allow us to identify the amino acid residue which is the hydrogen donor. We will study the products of the low temperature photochemistry. It is proposed that cryogenic conditions will improve the labelling efficiency. If successful this project will produce an important new method in photoaffinity labelling for identifying constituents of the active sites of biomolecues.

我们打算用不同的芳基叠氮化合物和 重氮化合物。纯修饰蛋白质的晶体将被光解 零下196度。我们将使用ESR波谱直接检测 生成的酶结合硝烯和卡宾。我们将使用ESR来测量 硝烯和卡宾与酶反应的动力学。我们 预计反应将涉及氢原子的量子转移 机械隧道形成自由基对。动能障碍将是 拟合到ECKART障碍物以获得障碍物宽度-移动距离 通过将氢从氨基酸残基隧道输送到卡宾或 是氮气。我们将通过ESR检测产物自由基对。的光谱 自由基对将给出该对分离的第二个衡量标准。 该对中存在的核超精细相互作用将使我们能够 确定作为氢供体的氨基酸残基。我们会研究 低温光化学的产物。现建议： 低温条件将提高标记效率。如果成功 该项目将产生一种重要的光亲和新方法。 用于鉴定生物分子活性部位成分的标记。