Ion-exchange properties of Cibacron Blue 3G-A Sepharose (Blue Sepharose) and the interaction of proteins with Cibacron Blue 3G-A.
The affinity for Blue Sepharose of several proteins of known structure showed a pH dependence governed by their isoelectric points; Blue Sepharose behaved like a strong cationic ion exchanger because of the negative charges of its dye ligand, Cibacron Blue. A study of the protein-Cibacron Blue interactions by phase partition and equilibrium dialysis revealed the presence of high-affinity binding sites both in the case of the (di)nucleotide-dependent enzymes that possess the structural domain known as "dinucleotide fold", and in the case of other proteins consisting almost entirely of alpha-helix (human haemoglobin, cytochrome c) or beta-sheet (human immunoglobulin G). The presence of additional sites of low affinity, probably situated at the protein surface, was also inferred from the equilibrium dialysis data. In some instances, in contrast with the Sepharose-immobilized dye, the interaction of free Cibacron Blue with proteins was not pH dependent. Steric factors could be responsible for such a differential behaviour. It is suggested that certain nucleotide-dependent enzymes might also bind to Blue Sepharose by ion exchange. Preparative applications of these findings are illustrated and discussed in terms of the optimization of affinity chromatography experiments.
D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange; D006452:Hemoglobinometry; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000032:analysis; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008929:Mitochondria, Heart / Q000201:enzymology; D009132:Muscles / Q000201:enzymology; D010047:Ovalbumin / Q000032:analysis; D011506:Proteins / Q000302:isolation & purification*; D011770:Pyruvate Kinase / Q000302:isolation & purification; D012685:Sepharose / Q000031:analogs & derivatives*; D013552:Swine; D014227:Triazines
D007074:Immunoglobulin G; D011506:Proteins; D014227:Triazines; C017842:cibacron blue-sepharose; C012842:Cibacron Blue F 3GA; D010047:Ovalbumin; D012685:Sepharose; D007770:L-Lactate Dehydrogenase; D011770:Pyruvate Kinase