Protein dye affinity chromatography using immobilized tetraiodofluorescein.

The chromatographic behavior of a heterogeneous protein mixture and of a series of homogeneous proteins on the immobilized dye tetraiodofluorescein has been observed and analyzed. Less than 6%, of the millimolar concentration of dye immobilized to a porous agarose matrix is accessible to protein. The affinity of a protein for immobilized dye is dramatically increased by insertion of apolar spacer atoms between the dye and the matrix. Dye columns constructed with a 9-atom spacer can be used to advantage for the retention and competitive elution of proteins not found previously amenable to dye chromatography.