TIME RESOLVED EVANESCENT WAVE-INDUCED FLUORESCENCE MEASUREMENTS OF SURFACE ADSORBED BOVINE SERUM-ALBUMIN

Despite intensive research, there are still many unresolved questions concerning the exact nature of protein binding at interfaces. One technique of growing interest for probing molecular interactions at surfaces is Time Resolved Evanescent Wave Induced Fluorescence Spectroscopy (TREWIFS), which combines the picosecond time resolution of time-correlated single photon counting, with the depth resolution provided by the use of an evanescent wave as excitation source. Using TREWIFS, the adsorption behavior of extrinsically labeled bovine serum albumin has been investigated as a function of penetration depth and relative surface protein concentration. We have observed changes in the emission kinetics of a protein-bound fluorescence probe upon adsorption at a hydrophilic surface, and find that these kinetics are highly dependent upon relative surface concentration of the labeled protein. Dye/protein complexes adsorbed at a hydrophobic surface exhibit a greater degree of fluorescence quenching than those at a hydrophilic one, due either to a higher surface coverage or to a closer-packed protein structure. © 1993 Academic Press, Inc.