Properties of concentrated macromolecular solutions

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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.