TIRF OF SALT AND SURFACE EFFECTS ON PROTEIN ADSORPTION

Total internal reflectance fluorescence (TIRF) spectroscopy was used to determine the kinetics of protein adsorption on modified (butylated or aminopropylated) quartz surfaces. The effects of surface characteristics and solution salt strength (concentration of NaCl or (NH4)2SO4) were studied on the rates of lysozyme and chymotrypsinogen A adsorption, with experimental data obtained in the form of fluorescence intensity as a function of time in response to a step input of protein. Since the observed changes in surface concentration reflect transport as well as kinetic limitations, kinetic data were extracted using a detailed model of the hydrodynamics and mass transfer in the TIRF flow cell. Because of uncertainty regarding constitutive relations for the adsorption process, the predictions of three different adsorption rate laws were compared in order to obtain bounds on the adsorption rate constant. Salt strength was found to have an appreciable effect on the adsorption rate, and the rate constant under a given set of conditions appeared to be correlated with the ultimate extent of adsorption observed under those conditions. © 1992.