Application of aqueous two-phase partition to the production of homogeneous preparations of fluorescently labelled human serum albumin

The development of a traceable molecular probe was investigated for the monitoring of partition behaviour of biomolecules in aqueous two-phase systems. This work was based upon the selective labelling of the free thiol group of human serum albumin (i.e. Cys34) with the fluorophore N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulphonic acid. The preparation of homogeneously labelled protein required purification operations. A succession of five processes was successfully applied, comprising two size-exclusion chromatographic operations by gel filtration and a series of three appropriately manipulated aqueous two-phase systems comprising PEG 1450 and phosphate salt. Aqueous two-phase partitioning is herein presented as an alternative to difficult separation and could be applied for ‘fine’ purifications.