Abstract
The effects of additions of low-molecular-mass salts on the properties of aqueous lysozyme solutions are examined by using the cloud-point temperature, $T_{cloud}$, measurements. Mixtures of protein, buffer, and simple salt in water are studied at pH=6.8 (phosphate buffer) and pH=4.6 (acetate buffer). We show that an addition of buffer in the amount above $I_{buffer} = 0.6$ mol dm$^{-3}$ does not affect the $T_{cloud}$ values. However, by replacing a certain amount of the buffer electrolyte by another salt, keeping the total ionic strength constant, we can significantly change the cloud-point temperature. All the salts de-stabilize the solution and the magnitude of the effect depends on the nature of the salt. Experimental results are analyzed within the framework of the one-component model, which treats the protein-protein interaction as highly directional and of short-range. We use this approach to predict the second virial coefficients, and liquid-liquid phase diagrams under conditions, where $T_{cloud}$ is determined experimentally.
Highlights
Taratuta et al [15] noticed that after a sufficient amount of buffer is added, Tcloud becomes insensitive to a further increase of ionic strength. This observation suggests that at certain ionic strength, the Coulomb interaction between proteins becomes sufficiently screened. We confirmed this finding for lysozyme in mixture with phosphate buffer and NaBr
In view of the new experimental results, shown in figure 3, we suggest this quantity to be correlated with the ionic strength of the added low-molecular-mass salt (Isalt) as follows: W(Isalt)/kB = a I salt + b
We present new measurements of the cloud-point temperature, Tcloud, for various lysozyme-buffersalt mixtures
Summary
Studies of the physico-chemical behaviour of mixtures of proteins and simple salts in water [1,2,3,4,5,6,7,8,9,10,11,12] are important to understand the stability of such mixtures and may yield improvements of methods for protein precipitation and crystallization [13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39]. The onset of the cloud-point temperature depends, on pH of the solution, and on the electrolyte concentration and its nature or composition if several salts are present For this reason, the Tcloud measurements represent a useful tool in the studies of salt-specific effects in protein solutions. For the chosen experimental method, the data collected in different laboratories may scatter much more than it is suggested by the precision of a single measurement One reason for this lies in the details of protein solution preparation, which appears to be more important than it is recognized by most of the researchers. The measurements were analyzed using the onecomponent model published recently [38] Based on this analysis and on our new Tcloud measurements, we predicted other thermodynamic quantities, including the full binodal curves and osmotic second virial coefficients for lysozyme in phosphate and acetate buffers in presence of low-molecular-mass salts
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