LOW TEMPERATURE CHEMISTRY/KINETICS/PHOTOAFFINITY LABELS

我们打算用各种芳基叠氮化物共价修饰α-胰凝乳蛋白酶， 重氮化合物。 纯改性蛋白质的晶体将被光解 零下196度 我们将使用ESR光谱直接检测 生成的酶结合氮烯和卡宾。 我们将使用ESR来测量 氮烯和卡宾与酶反应的动力学。 我们 预计该反应将涉及氢原子通过量子转移 机械隧穿以给出一个基对。 动能屏障将是 适合Eckart屏障，以获得屏障宽度-行驶距离 通过将氢从氨基酸残基隧穿到卡宾，或 氮烯。 我们将通过ESR检测产物自由基对。 的光谱 根对将给出对的分离的第二度量。 原子核中的超精细相互作用将使我们能够 鉴定作为氢供体的氨基酸残基。 我们将研究 低温光化学的产物。 拟将 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新方法， 用于识别生物分子活性位点成分的标记。

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.