NONCOVALENT INTERMOLECULAR INTERACTIONS IN BIOCHEMISTRY

生物化学中的非共价分子间相互作用

• 批准号: 6105119
• 负责人: Allen P Minton
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6105119
• 关键词: analytical method; biomedical equipment development; chemical kinetics; fiber optics; intermolecular interaction; macromolecule; mathematical model; membrane transport proteins; method development; proteins; receptor binding; sedimentation equilibrium; spectrometry; tryptophan synthase

Statistical thermodynamic and kinetic models have been developed to describe rates and equilibria of reversible adsorption of proteins to surfaces under conditions such that a particular protein may adsorb in more than one conformation. The results of kinetic simulations suggest that early binding of conformations that are energetically favored at low surface occupancy, but ultimately entropically disfavored at high surface occupancy, may result in a substantial kinetic barrier to the attainment of equilibrium. It follows that under conditions that would ultimately lead to high surface occupancy at equilibrium, conventional measurements of the kinetics of adsorption may fail to reveal essential aspects of the process occurring on a time scale several orders of magnitude longer than that characterizing the initial low-occupancy (rapid) phase of adsorption. Studies aimed at physico-chemical characterization of poly-amido-amine (PAMAM) dendritic polymers in aqueous solution were initiated. These highly compact, homogeneous polymers may be synthesized to approximately the same size and shape as globular proteins, and their surface residues may be modified (for example, hydroxylated) to minimize reactivity with biopolymers. Such polymers may thus serve as models for the volume-excluding properties of proteins in highly concentrated solution. Measurements have been made of the absorbance spectrum, density increment, refractive increment, sedimentation coefficient, diffusion coefficient, buoyant molar mass (direct measurement) and actual molar mass (indirect measurement). Purity and homogeneity of the polymer were monitored via high-performance size exclusion chromatography. Measurements of intermolecular interaction via sedimentation equilibrium are in progress. A spectrophotometric absorbance scanner based has been designed and built, the purpose of which is to acquire UV-visible absorbance data as a function of both wavelength and radial position from solutions of one or more macromolecular solutes that have been centrifuged to sedimentation equilibrium. The instrument is currently being tested and the design undergoing modification as necessary. Preliminary results suggest the instrument will be capable of providing data of substantially higher spectrophotometric accuracy, precision and spatial resolution than attainable via currently available instrumentation. The technique of tracer sedimentation equilibrium developed earlier in our laboratory has been extended both theoretically and experimentally to permit the thermodynamic activity and/or state of association of a trace amount of labelled macrosolute to be characterized in the presence of an arbitrary concentration of a second macrosolute. Using this technique the thermodynamic activity of dilute fibrinogen (Fbg) has been measured in solutions of bovine serum albumin (BSA) at concentrations of up to 100 mg/ml. It was found that at a BSA concentration of 80 mg/ml, which is comparable to the total concentration of protein in blood serum, the thermodynamic activity of Fbg is ten-fold greater than its concentration, suggesting that the affinity of Fbg for platelet receptors in blood serum may be substantially larger than suggested by binding experiments carried out in vitro. The presence of a substantial population of soluble oligomers of Fbg has been demonstrated in the presence of Ca++ and moderate concentrations (<40 mg/ml) of BSA.

统计热力学和动力学模型 被开发来描述可逆的速率和平衡 在一定条件下蛋白质吸附到表面 特定的蛋白质可以以不止一种构象吸附。这 动力学模拟的结果表明，早期结合 在低表面上有利于能量的构象 占用率，但最终在高表面处熵不利 占用，可能会导致对 达到平衡。由此可见，在以下条件下 最终会导致平衡时的高表面占有率， 吸附动力学的常规测量可能无法 揭示在时间尺度上发生的过程的基本方面 比特征值长几个数量级 吸附的初始低占有率（快速）阶段。 研究旨在 聚酰胺基胺 (PAMAM) 的物理化学表征 引发水溶液中的树枝状聚合物。这些高度 可以合成致密、均质的聚合物 与球状蛋白质的大小和形状大致相同，并且 它们的表面残基可能被修饰（例如羟基化） 以尽量减少与生物聚合物的反应。因此，此类聚合物可以 作为蛋白质体积排除特性的模型 高浓度溶液。已进行测量 吸收光谱、密度增量、折射率增量、 沉降系数、扩散系数、浮力摩尔质量 （直接测量）和实际摩尔质量（间接测量） 测量）。聚合物的纯度和均质性 通过高性能尺寸排阻色谱法进行监测。 通过沉降测量分子间相互作用 平衡正在进行中。 分光光度法吸光度 基于扫描仪的设计和构建，其目的是 获取紫外-可见光吸光度数据作为两者的函数 来自一种或多种溶液的波长和径向位置 经离心沉淀的大分子溶质 平衡。该仪器目前正在测试和设计中 进行必要的修改。初步结果表明 该仪器将能够提供大量数据 更高的分光光度准确度、精度和空间 分辨率高于当前可用仪器所能达到的分辨率。 发展较早的示踪沉降平衡技术 我们实验室的理论和实践都得到了扩展 通过实验允许热力学活动和/或状态 微量标记的大溶质的关联 特征在于存在任意浓度的 第二大溶质。使用该技术，热力学 在以下溶液中测量了稀释纤维蛋白原 (Fbg) 的活性 浓度高达 100 mg/ml 的牛血清白蛋白 (BSA)。 结果发现，BSA 浓度为 80 mg/ml 时， 与血清中蛋白质的总浓度相当， Fbg的热力学活性比其高十倍 浓度，表明 Fbg 对血小板的亲和力 血清中的受体可能比建议的要大得多 通过在体外进行的结合实验。存在一个 大量可溶性 Fbg 寡聚体已被 在存在 Ca++ 和中等浓度的情况下得到证明 (<40 毫克/毫升) BSA。