Chromatography of proteins on columns of polyvinylpolypyrrolidone using adsorbed textile dyes as affinity ligands.
A simple and inexpensive chromatography system for proteins is introduced. When the amino derivatives of chlorotriazine dyes or other azo dyes were added to an aqueous slurry of the crosslinked polymer polyvinylpolypyrrolidone they were adsorbed, thus forming an immobilized dye chromatographic matrix. The association of the textile dyes with polyvinylpolypyrrolidone did not prevent them from acting as affinity ligands for proteins. Parameters such as ionic strength, dye concentration, and column size modulated the affinity effect exerted by the immobilized dyes. Lysozyme present in an egg white protein mixture bound to a column onto which the amino derivative of Procion Brown H-A was adsorbed and was eluted with a linear gradient of KCl. The resulting purification of the enzyme was 37-fold with 80% of the original activity being recovered. Free dye eluting with the lysozyme was removed on a column of polyvinylpolypyrrolidone equilibrated with 0.5 M KCl. After chromatography, the dye column was regenerated with 0.5 M NaOH and recharged with dye. The system presented here allows one to initially screen large numbers of potentially useful protein ligands to optimize a protein separation, followed by scaleup to a system size determined by the user.
Giuliano
K A
KA
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
D000327:Adsorption; D001426:Bacterial Proteins / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods; D004396:Coloring Agents; D003207:Conalbumin / Q000302:isolation & purification; D004527:Egg Proteins / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007202:Indicators and Reagents; D008024:Ligands; D008970:Molecular Weight; D009113:Muramidase / Q000302:isolation & purification; D011205:Povidone; D011506:Proteins / Q000302:isolation & purification*
D001426:Bacterial Proteins; D004396:Coloring Agents; D004527:Egg Proteins; D007202:Indicators and Reagents; D008024:Ligands; D011506:Proteins; D003207:Conalbumin; D009113:Muramidase; D011205:Povidone