Immobilization of proteins via arginine residues.
A new method for activating polyacrylamide beads to bind proteins via arginine residues is described. The linking reagent, 4-(oxyacetyl)phenoxyacetic acid (OAPA), has been synthesized and characterized. OAPA reacts with arginine or N alpha-acetyl-L-arginine with a stoichiometry of 2 to 1. As expected for an arginine-specific reagent, OAPA inactivates horse liver alcohol dehydrogenase in a time-dependent manner, with the rate of this inactivation decreasing sixfold in the presence of 1 mM NADH. The presence of the carboxyl group in the linking reagent allows efficient coupling to aminated polyacrylamide beads. These derivatized beads are capable of binding various proteins via arginine residues in a time- and pH-dependent manner. Capacities range from less than 0.5 mg/ml to greater than 11 mg/ml, depending on the protein. The proteins are bound in a stable linkage, and preblocking the beads with either arginine or N alpha-acetyl-L-arginine eliminates all protein binding. Preblocking of the protein ubiquitin with OAPA reduces binding to a level compatible with the amount of underivatized ubiquitin remaining. The specificity, water solubility, negative charge, and linking ability of OAPA make it an especially valuable tool, both as a protein-modification reagent and as a linking reagent in preparing specialized affinity chromatographic media.
D000180:Acrylic Resins; D000426:Alcohol Dehydrogenase / Q000037:antagonists & inhibitors; D000818:Animals; D001120:Arginine; D002846:Chromatography, Affinity / Q000379:methods*; D003432:Cross-Linking Reagents; D006016:Glycolates / Q000138:chemical synthesis; D006736:Horses; D008099:Liver / Q000201:enzymology; D011485:Protein Binding; D011506:Proteins; D013056:Spectrophotometry, Ultraviolet; D014452:Ubiquitins; D000099166:Polyacrylamides
D000180:Acrylic Resins; D000426:Alcohol Dehydrogenase; D001120:Arginine; D003432:Cross-Linking Reagents; D006016:Glycolates; D011506:Proteins; D014452:Ubiquitins; D000099166:Polyacrylamides; C052188:4-(oxoacetyl)phenoxyacetic acid