Interaction of the dye Remazol Yellow GGL to prealbumin and albumin studied by affinity phase partition, difference spectroscopy and equilibrium dialysis.
In partition experiments in aqueous two-phase systems composed of 10% (w/w) dextran (Mr = 500,000) and 7.5% (w/w) poly(ethylene-glycol) (Mr = 6,000) prealbumin and albumin are directed into the dextran-rich phase. Addition of Remazol Yellow GGL covalently bound to poly(ethylene-glycol) causes a transfer of prealbumin and albumin into the poly(ethylene-glycol)-rich phase. This indicates an interaction of both proteins with the dye (affinity phase partitioning). The affinity partitioning effect on prealbumin is markedly increased by an excess of monomeric albumin. This points to an interaction of the two proteins in the presence of the dye. Binding of free Remazol Yellow GGL to prealbumin and albumin was investigated by means of equilibrium dialysis and difference spectroscopy. In respect to prealbumin equilibrium dialysis resulted in the binding of four molecules of the dye to two classes of binding sites with dissociation constants of KH = 3.3 microM and KL = 258 microM respectively whereas albumin was found to bind eight molecules of the dye to two classes of binding sites with KH = 5.8 microM and KL = 282 microM. Similar binding stoichiometries were found by difference spectroscopy. By application of difference spectroscopy and affinity phase partitioning thyroxine and triiodothyronine known as natural ligands of prealbumin and albumin were found to compete with Remazol Yellow GGL for the dye binding sites of the proteins.
D000345:Affinity Labels; D000418:Albumins / Q000032:analysis*; D001391:Azo Compounds; D001667:Binding, Competitive; D003956:Dialysis / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011228:Prealbumin / Q000032:analysis*; D011485:Protein Binding; D012995:Solubility; D013057:Spectrum Analysis / Q000379:methods; D013974:Thyroxine / Q000032:analysis; D014284:Triiodothyronine / Q000032:analysis
D000345:Affinity Labels; D000418:Albumins; D001391:Azo Compounds; D011228:Prealbumin; D014284:Triiodothyronine; C035187:reactive yellow 13; D013974:Thyroxine