Application of total internal reflection fluorescence to probe surface diffusion and orientation of adsorbed proteins

In the first part of this dissertation, total internal reflection fluorescence (TIRF) is employed simultaneously with fluorescence recovery after pattern photobleaching (FRAPP) to measure surface self diffusion coefficients and mobile fractions of fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (BSA) and bovine pancreatic ribonuclease A (RNase) irreversibly adsorbed to spin-cast polymeric substrates. The relative quantum yield for FITC-RNase was found experimentally to depend on fluorophore concentration due to fluorescence concentration quenching. The fluorophore concentration dependence of the relative quantum yield was determined experimentally for RNase adsorbed to both polystyrene and polydimethylsiloxane (PDMS) surfaces using TIRF data. The functional dependence of the relative quantum yield was then used, and a modified data analysis procedure developed, to correctly analyze FRAPP experiments when the relative quantum yield is not a constant. Theoretical calculations show that if not properly taken into account, the presence of concentration quenching will lead to overestimated values of the mobile fraction and, for certain fluorescence recovery experimental configurations, the diffusion coefficient is overestimated as well.;In the second part of this dissertation TIRF, reflectometry and fluorescence spectroscopy are used to show that FITC-labeled hen egg-white lysozyme undergoes a packing-induced reorientation as it adsorbs to negatively charged silica surfaces. Fluorometric titrations of FITC-lysozyme indicate that the conjugated fluorescein has an apparent pK...