CORRELATIONS FOR THE PARTITION BEHAVIOR OF PROTEINS IN AQUEOUS 2-PHASE SYSTEMS - EFFECT OF OVERALL PROTEIN-CONCENTRATION

The effect of protein concentration in partitioning in PEG/ salt aqueo us two-phase systems has been investigated. PEG 4000/phosphate systems in the presence of 0% w/w and 8.8% w/w NaCl have been evaluated using amyloglucosidase, subtilisin, and trypsin inhibitor. Also, a PEG 4000 /phosphate system with 3% w/w NaCl was used for alpha-amylase. The con centration of the protein in each of the phases affected its partition behavior. The pattern for the individual proteins was dependent on th eir physicochemical properties. In the top phase, maximum protein conc entration was determined mainly by a steric exclusion effect of PEG, a nd hydrophobic interaction between PEG and proteins. In the bottom pha se, maximum concentration was determined mainly by a salting-out effec t of the salts present. As the ionic strength was increased in the sys tems the concentration in the top phase increased for all proteins. In the bottom phase an increase in ionic strength increased the salting- out effect. Amyloglucosidase had a very low maximum concentration in t he PEG-rich top phase which was probably due to its large size (steric exclusion) and low hydrophobicity, and a high concentration in the sa lt-rich bottom phase due to its high hydrophilicity. In the case of su btilisin and trypsin inhibitor, their high concentrations in the top p hase were due to their hydrophobic nature (hydrophobic interaction wit h PEG) and small size (negligible steric exclusion). The maximum conce ntration in the bottom phase for trypsin inhibitor was lower than that of subtilisin which was probably due to its higher hydrophobicity and , hence, a stronger salting-out effect. The protein concentration in e ach of the two phases was correlated with a ''saturation''-type equati on. The partition coefficient could be satisfactorily predicted, as a function of the overall protein concentration, by the ratio between th e ''saturation'' equations of the two individual phases. Better correl ations were obtained when an empirical sigmoidal Boltzmann equation wa s fitted to the data, since in virtually all cases the partition coeff icient is constant at low protein concentration (true partitioning) an d changes to a different constant value at a high overall protein conc entration. (C) 1996 John Wiley & Sons, Inc.