HYDROPHOBIC PARTITIONING OF PROTEINS IN AQUEOUS 2-PHASE SYSTEMS

The hydrophobicity of five proteins was estimated by reversed-phase ch romatography (RPC), hydrophobic-interaction chromatography (HIC), and precipitation with ammonium sulfate. These data were correlated to par tition behavior in aqueous two-phase systems. A parameter (1/m) that was derived from the precipitation curves is based on the solubility o f proteins in an electrolyte solution. The correlation between 1/m an d the retention times in HIC and RPC was poor due to interaction effec ts with the chromatography matrices and probably partial unfolding of the proteins; however this parameter (1/m) was expected to be a measu re of hydrophobicity of proteins that relates better than the chromato graphic data to experiments where the hydrophobic behavior of proteins in an aqueous solution is used for their separation. The partition. b ehavior of the five proteins in aqueous two-phase systems (ATPS) in th e absence and presence of NaCl was investigated. A poor correlation wa s found between log K (K is the partition coefficient) in ATPS and the hydrophobicity values measured by RPC and HIC; however a very good co rrelation was found between log 1/m which is a measure of protein hyd rophobicity based on the solubility of the protein during precipitatio n and log K, particularly in PEG/PO4 systems with added NaCl. The para meter (1/m) also demonstrated a good correlation with log K in PEG/de xtran systems. A simple correlation for the prediction of partitioning in specific ATPS based on this parameter has been evaluated. An expre ssion describing its resolution power R, and a parameter describing th e hydrophobicity of the system, P-o, was determined making the correla tion potentially predictive for other proteins in the ATPSs used. Hydr ophobicity of proteins was better exploited in PEG/PO4 systems than in PEG/dextran ones as a much higher resolution (R) is obtained in the f ormer. (C) 1996 by Elsevier Science Inc.