SALT-INDUCED IMMOBILIZATION OF PROTEINS ON A HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC EPOXIDE AFFINITY SUPPORT

The immobilization of thirteen proteins on a high-performance liquid c hromatography epoxy affinity support was studied as a function of ammo nium sulfate concentration. Without exception the fraction of protein immobilized at 20 h increased with increasing salt concentration at hi gher salt concentrations. Ten of the proteins were 95-100% immobilized in 20 h at ammonium sulfate concentrations ranging from 0.4 to 2.5 M. At lower salt concentrations the affinity support exhibited lower rea ctivity with all proteins. A kinetic model is proposed in which protei n in solution is at equilibrium with a noncovalent protein-affinity su pport complex. The nucleophiles on the protein of the complex then rea ct in a slow step with epoxy groups on the affinity phase. The depende nce of immobilization on salt concentration is interpreted as a salt i nduced hydrophobic interaction on the equilibrium formation of the non covalent protein-affinity matrix complex.