We have introduced an experimental method for automatically and rapidly performing a measurement of the concentration dependence of light scattering over a wide range of concentrations. This method allows the maximum amount of information to be extracted from the concentration dependence of light scattering, due to the acquisition of data at equal logarithmic increments of concentration, so that data obtained at both very low and very high concentration receive equal statistical weight [5
The method has been validated by measuring the concentration dependence of three proteins, BSA, ovalbumin and ovomucoid. It is shown that the concentration dependence of each may be accounted for quantitatively over a wide concentration range by a simple model, according to which each protein molecule interacts with other protein molecules only via short-ranged repulsive interactions that may be approximated as hard particle interactions, and does not self-associate to any significant extent over the concentration range examined. Molecular weights obtained for all three proteins agree well with literature values [4
Our finding that the concentration dependence of scattering for BSA at high concentration may be accounted for by an effective hard sphere model without significant self-association at concentrations below 100 g/L agrees well with the results of earlier measurements of the light scattering, osmotic pressure and sedimentation equilibrium of concentrated BSA solutions [12
]. Our finding that the concentration dependence of the scattering of ovalbumin may be accounted for by an effective hard sphere model without significant self-association at concentrations below 130 g/L agrees with a previous interpretation of the concentration dependence of osmotic pressure [19
]. However, an earlier measurement of sedimentation equilibrium of concentrated ovalbumin [20
] was interpreted as evidence for equilibrium monomer-trimer or monomer-dimer-tetramer self-association. The automated dilution system developed here provides a significant improvement in the experimental precision of measurement over a wide range of concentration, especially at high concentration. Attempts to fit our data to a non-ideal monomer/ trimer or monomer/ dimer/ tetramer model [5
] were unsuccessful. Hence the origin of the discrepancy between light scattering and sedimentation equilibrium results remains unresolved. We are unaware of previous characterizations of the colligative properties of ovomucoid at high concentration, although it has been used as an “inert” cosolute at high concentration to study the quantitative effect of macromolecular crowding on protein aggregation [21
Analytical methods that are used to characterize proteins in solution generally require dilution to solvent conditions that are very different than those in which the protein functions in vivo
] or in a biopharmaceutical formulation [22
]. In contrast, the apparatus and experimental technique method introduced here provide a relatively inexpensive and moderately high throughput tool for precise quantitative characterization of the state of association of proteins in solution at all concentrations up to the solubility limit. In a following paper we shall describe how data derived from measurements like those described here may be used to detect and quantitatively characterize weak equilibrium self-associations in a highly nonideal solution.
The new method presented here may be compared to the best current automated method for measuring the concentration dependence of light scattering in solutions of proteins and other macromolecules, in which a stock protein solution is automatically mixed with buffer in varying ratios prior to introduction into light scattering and concentration detection flow cells [14
]. The method presented here is easier to implement for very concentrated protein solutions, as no transport of concentrated solution through narrow bore tubing or filters is required. Moreover, no separate concentration flow detector is needed, since the protein solution being measured remains in the measurement cell throughout and the extent of dilution is determined precisely by the high resolution stepping motor-controlled syringe pumps.