NONCOVALENT INTERMOLECULAR INTERACTIONS IN BIOCHEMISTRY

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， 蛋白质在一定条件下吸附到表面上， 特定蛋白质可以以一种以上构象吸附。的 动力学模拟的结果表明， 在低表面上能量有利的构象 占据，但最终在高表面熵不利 占用，可能会导致一个实质性的动力障碍， 达到平衡。因此，在以下条件下， 将最终导致平衡时的高表面占有率， 吸附动力学的常规测量可能不能 揭示在时间尺度上发生的过程的基本方面 几个数量级长的特征， 初始低占有（快速）吸附阶段。 研讨会等 聚酰胺-胺（PAMAM）的理化表征 引发水溶液中的树枝状聚合物。这些高度 可以合成致密、均匀的聚合物， 与球状蛋白质的大小和形状大致相同， 它们的表面残基可以被修饰（例如，羟基化）， 以最小化与生物聚合物的反应性。这样的聚合物因此可以 作为蛋白质体积排除特性的模型， 高度浓缩的溶液。测量了 吸收光谱，密度增量，折射增量， 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）。聚合物的纯度和均匀性是 通过高效尺寸排阻色谱法监测。 通过沉降测量分子间相互作用 平衡正在进行中。 A分光光度法吸光度 扫描仪的基础上已经设计和建造，其目的是 以获取作为两者的函数的UV-可见光吸光度数据， 波长和径向位置从一个或多个 已离心沉淀的大分子溶质 均衡该仪器目前正在测试中， 进行必要的修改。初步结果表明 该仪器将能够提供基本上 更高的分光光度准确度、精度和空间 比通过目前可用的仪器可达到的分辨率。 示踪沉降平衡技术发展较早 在我们的实验室里，理论上和 实验上允许的热力学活动和/或状态 痕量的标记大溶质的缔合， 其特征在于存在任意浓度的 第二大溶质。利用这种技术， 已在以下溶液中测量了稀释的纤维蛋白原（Fbg）的活性： 牛血清白蛋白（BSA），浓度高达100 mg/ml。 发现在80 mg/ml的BSA浓度下， 与血清中蛋白质的总浓度相比， Fbg的热力学活性比其 Fbg对血小板的亲和力 血清中的受体可能比建议的大得多 通过在体外进行的结合实验。的存在 大量的Fbg可溶性低聚物已经被 在存在Ca++和中等浓度的情况下证明 （<40 mg/ml）BSA。

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.