Buffer Effect on Protein Adsorption at Liquid/Solid Interface

This work shows buffer choice and buffer concentration can drastically affect protein adsorption. We used ATR/FTIR to compare the adsorption kinetics and secondary structural evolution of BSA, IgG, fibrinogen and lysozyme on a Ge surface, buffered in phosphate buffered saline (PBS) and tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) at pH 7.4. The adsorption behaviors of the four proteins are multilayer in nature, and all exhibit a short period of rapid initial adsorption with larger secondary structural changes, followed by a long period of quasi-linear adsorption. For BSA, IgG and fibrinogen, PBS buffer depresses the adsorption in the quasi-linear kinetic region compared with Tris-HCl buffer, whereas lysozyme adsorption is relatively insensitive to buffer choice. Buffer concentration also affects protein adsorption. BSA adsorption increases monotonically with Tris-HCl concentration while the variation with PBS concentration is nonmonotonic. The secondary structure in the adsorbed phase is in general quite different from that in the bulk solution; however, buffer choice does not have a significant effect on secondary structural evolution of adsorbed proteins especially when comparisons are made on the basis of adsorbed amount. Although PBS is the most commonly used buffer at physiological pH, the role of phosphate ions in PBS buffer and their effect on protein adsorption are rather complex because phosphate ions adsorb competitively with protein molecules and more than one type of phosphate ions can exist both in the bulk solution and in the adsorbed phase. The competitive nature of protein and phosphate ion adsorption is demonstrated for IgG adsorption in a sequenced adsorption/flushing/displacement experiment.