Polymer–protein interactions in aqueous two phase systems: fluorescent studies of the partition behaviour of human serum albumin

This study describes the partitioning of fluorescent macromolecules in aqueous two-phase systems (ATPS) comprising phosphate salt and poly(ethylene glycol) of three different molecular masses (i.e. 1000, 1450 and 2000 Da). The impact of system assembly was studied with fluorescent macromolecules introduced in contact with either (i) first salt, then polymer or (ii) first polymer, then salt, or (iii) with both salt and polymer simultaneously. Native human serum albumin (HSA) and derivatives labelled with N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulphonic acid (1,5-IAEDANS) were partitioned using selected ATPS. Partitioning behaviour was characterised by molecular rotational studies of recovered proteins based upon changes of depolarisation. Measurements were undertaken by steady-state fluorescence or time-decay fluorescence using a single-photon counting system. In addition, circular dichroism was used as a tool for the study of macromolecular secondary structure. Two discrete categories of stable molecular structure have been identified that exist irrespective of the phase environment. The findings form the basis for a discussion of polymer–protein interactions and the molecular micro-environment of proteins in ATPS.