REACTIONS CATALYZED BY ENZYMES ENTRAPPED IN GEL GLASSES

说明(改编自申请)：建议的基本概念 研究是为了调查影响催化反应的因素 固定在溶胶-凝胶玻璃中的酶。尽管有数量巨大的 发挥重要的和有充分证据的生理功能的蛋白质 令人惊讶的是，它们的治疗特性几乎没有被利用， 原因是在许多情况下，它们是敏感的，化学上不稳定。在……里面 特别是，包埋在溶胶-凝胶材料中的蛋白质可以用来开发 用于检测代谢物的生物传感器，因此在 开发新的临床分析方法。磷脂酶A2(PLA2) 代表了研究多孔介质中酶反应的理想模型，因为 这些蛋白质很小，很硬，很稳定，可以催化一类初级的 反应(水解)。调查人员将蟒蛇固定住 硅胶中的鱼磷脂酶A2，并观察到 3-辛酰氧基-4-硝基苯甲酸(ONB)的水解 在硝基苯酚离子的可见光区。这种水解反应将 在考虑实验因素的情况下测试活动的变化 例如老化/结构演变、储存条件、温度、干燥 极性、pH、不同底物的可及性、浓度 不同的二价金属，以及酶的构象状态。至 了解分子和离子的扩散特性 硅溶胶-凝胶的空隙是他们计划解决的一个方面。他们 已经捕获了有机荧光染料并观察到了 当整体被放入时，荧光信号作为时间的函数 接触淬火剂的溶液，如丙烯酰胺。因为PLA2是 依赖钙的酶，二价金属的扩散特性，如 将研究Ca2、Mg2和Zn2对金属离子荧光探针的包埋作用。 蛋白质的构象状态将通过荧光法进行评估。 测量。使用O2、I-和丙烯酰胺等淬火剂将提供 关于不同芳香族氨基酸的可及性信息和 关于蛋白质的三元结构。荧光的最大值 将使用谱以及谱的半峰值的全宽度 将不同组的色氨酸联系起来。荧光寿命 还将利用测量来识别不同的色氨酸残留物。

Description (Adapted from Application): The fundamental concept of the proposed research is to investigate the factors that affect reactions catalyzed by enzymes immobilized in sol-gel glasses. Although there is an immense number of proteins that perform important and well-documented physiological functions, astonishingly little use has been made of their therapeutic properties, the reason being in many cases that they are sensitive and chemically unstable. In particular, proteins entrapped in sol-gel materials can be used to develop biosensors for the detection of metabolites, thereby being promising for the development of novel clinical analytical methods. Phospholipases A2 (PLA2) represent ideal models to study enzymatic reactions in porous media because these proteins are small, rigid and stable, and catalyze an elementary class of reactions (hydrolysis). The investigators immobilized Agkistrodom piscivorus piscivorus phospholipase A2 in silica sol-gels and have observed the hydrolysis of 3-octonoyloxy-4-nitrobenzoic acid (ONB), following the appearance in the visible region of the nitrophenolate ion. This hydrolysis reaction will be carried out to test changes in activity, considering experimental factors such as aging/structural evolution, storage conditions, temperature, drying, polarity, pH, the accessibility of different substrates, concentration of different divalent metals, and conformational states of the enzyme. To understand the diffusion properties of molecules and ions through the interstices of the silica sol-gel is an aspect that they plan to address. They have entrapped organic fluorescent dyes and have observed the attenuation of the fluorescence signal as a function of time when the monoliths are put in contact with solutions of a quencher such as acrylamide. Since PLA2s are calcium-dependent enzymes, the diffusion properties of divalent metals such as Ca2+, Mg2+ and Zn2+ will be studied entrapping metal ion fluorescent probes. The conformational states of the protein will be assessed from fluorometric measurements. The use of quenchers such as O2, I-, and acrylamide will provide information about the accessibility of the different aromatic amino acids and about the ternary structure of the protein. The maxima of the fluorescence spectra along with the full width at half maximum of the spectra will be used to correlate different groups of tryptophans. Fluorescence lifetime measurements will also be employed to identify different tryptophil residues.