TIRF of salt and surface effects on protein adsorption: I. Equilibrium

Abstract

Total internal reflectance fluorescence spectroscopy was used to study protein adsorption under conditions relevant to those used in liquid chromatography, particularly hydrophobic interaction chromatography. The effects of surface characteristics (butylated or aminopropylated) and solution salt conditions (concentration of NaCl or (NH 4) 2SO 4) on the extent of adsorption of lysozyme and chymotrypsinogen A on modified quartz surfaces were studied. An internal calibration technique was used to relate the intrinsic fluorescence intensity of the proteins to surface concentration. The two proteins displayed similar adsorption characteristics. Adsorption was found to be largely irreversible on the butylated slides but more readily reversible on the aminopropylated slides. Both proteins adsorbed on aminopropylated surfaces despite the expected electrostatic repulsion between the positively charged surface and the proteins, both of which bear a net positive charge at pH 7. Salt strength affected the extent of surface coverage, even in the plateau regions of adsorption isotherms; thus monolayer coverage was not attained under all conditions at high bulk protein concentrations. Salt effects on adsorption on butylated surfaces appeared to be due largely to those on solubility, but on aminopropylated surfaces the interaction of salt with the charged surface was apparent in adsorption behavior.