Properties of concentrated macromolecular solutions

1.利用本实验室开发的一种技术和仪器，测量了总浓度高达100g/L的三种蛋白质(牛血清白蛋白、卵白蛋白和类卵粘蛋白)与小分子渗透剂三甲胺-N-氧化物(TMAO)混合物的光散射强度随组成的变化。用本实验室发展的适用于非缔合溶质的二元混合物的公式对结果进行了分析，这些混合物在高浓度下表现出排斥的自作用和异作用。结果可以用一个半经验模型来定量解释，在该模型中，蛋白质被表示为一个球体，其有效体积取决于它是与自身还是与TMAO相互作用，而TMAO被表示为一个较小的恒定体积的球体。这个模型的基本原理是，在测量条件下，这里研究的每个蛋白质都带有净负电荷。因此，蛋白质分子之间的自身相互作用具有排斥静电相互作用的元素，而蛋白质分子和中性TMAO分子之间的相互作用在很大程度上是没有的。 2.利用本实验室开发的非理想示踪沉淀平衡技术和仪器，在总浓度高达100g/L、温度在5-37摄氏度的条件下，研究了三种稀示物蛋白(牛血清白蛋白、卵清蛋白和超氧化物歧化酶)和两种浓缩蛋白(牛血清白蛋白和卵白蛋白)在总浓度高达100g/100g/100g/100g/100g/100g/100g/100g/100g/100g/100g/100g/100g/100g/100g/100g/100g/100g/100g/100g/100g/100g/100g/100g，温度5-37℃的条件下，三种稀释示踪蛋白(牛血清白蛋白、卵清蛋白和超氧化物歧化酶)和两种浓缩蛋白(牛血清白蛋白和卵白蛋白)的混合物结果可以用半经验模型来定量解释，根据该模型，每个蛋白质被表示为一个球体，浓缩蛋白质的自相互作用纯粹是排斥的，浓缩和稀释蛋白质的异相互作用主要是排斥的，但由于一个小的吸引相互作用，排斥的后果略有减少，可以表征为极弱的异结合作用(Kd约为10 mm)。在实验不确定度范围内，排斥性相互作用被发现与TO无关，并且只有一种有吸引力的相互作用，即示踪剂SOD和卵粘蛋白之间的相互作用，在所研究的范围内表现出很小但显著的温度依赖性。 3.与德国Rivas(CIB，马德里)合作，就高度非理想溶液中的沉积平衡问题撰写了一篇简短的评论。本文介绍了定量解释数据的实验方法和一般理论。

1. The composition dependence of the light scattering intensity of mixtures of each of three proteins, BSA, ovalbumin, and ovomucoid, and a small molecule osmolyte, trimethylamine-N-oxide (TMAO) at total concentrations of up to 100 g/l was measured using a technique and instrumentation developed in this laboratory and described in previous reports. The results were analyzed using formalism developed in this laboratory appropriate for binary mixtures of non-associating solutes exhibiting repulsive self- and hetero-interaction at high concentration. The results could be quantitatively accounted for by a semiempirical model in which the protein was represented as a sphere, the effective volume of which depended upon whether it was interacting with itself or with TMAO, and TMAO was represented as a smaller sphere of constant volume. The rationale for this model is that at under the conditions of measurement, each of the proteins studied here bears a net negative charge. Thus self-interactions between protein molecules have an element of repulsive electrostatic interaction that is largely absent from the interaction between a protein molecule and a neutral TMAO molecule. 2. The composition and temperature dependence of the sedimentation equilibrium of mixtures of each of three dilute tracer proteins (BSA, ovalbumin, and superoxide dismutase or SOD) and and two concentrated proteins (BSA and ovalbumin) at total concentrations of up to 100 g/l and temperatures between 5 and 37 degrees Centigrade, by means of a technique (nonideal tracer sedimentation equilibrium) and instrumentation developed in this laboratory, described in previous reports. The results could be quantitatively accounted for by semiempirical models, according to which each protein was represented as a sphere, self-interaction of concentrated protein was purely repulsive, and hetero-interaction of concentrated and dilute protein was primarily repulsive, but the consequences of repulsion reduced slightly due to a small attractive interaction that could be characterized as extremely weak heteroassociation (Kd of order 10 mM). The repulsive interactions were found to be independent of to within experimental uncertainty, and only one attractive interaction, that between tracer SOD and ovomucoid, exhibited a small but significant temperature dependence over the range studied. 3. In collaboration with German Rivas (CIB, Madrid) a mini-review on the subject of sedimentation equilibrium in highly nonideal solutions was written. The review describes experimental methods and general theory for quantitative interpretation of data.