On the precipitation of proteins by polymers: The hemoglobin—polyethylene glycol system

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Abstract
The addition of polyethylene glycol (PEG) of MW 6000 to solutions of oxy‐ and deoxyhemoglobins results in an increase in the thermodynamic activity of these proteins. This in turn results, when PEG concentration is high enough, in phase separation into two phases; a protein‐rich, PEG‐poor phase and a PEG‐rich, protein‐poor phase. With increasing PEG concentration, the protein‐rich amorphous phase becomes metastable and is converted into a well‐defined crystalline or polymer phase. The logarithm of protein solubility is a linear function of PEG content up to a protein concentration of 150 g/L because the expression for the activity coefficient can, up to this concentration range, be approximated by a logarithmic function. Curvature appears at higher protein concentrations. Activities obtained by extrapolation from linear portions of the function, showing an unchanged, well‐defined crystalline state, yield an activity coefficient for the saturated PEG‐free protein solution in agreement with the appropriate values obtained from hard‐sphere calculations of excluded volume [Ross, P. D. & Minton, A. P. (1977) J. Mol. Biol. 112, 437–452]. Solutions containing two hemoglobin species showed cocrystallization of the hemoglobins with a triple point where two crystal forms can be shown to coexist.

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