Studies on Electrostatic Interactions between Biomolecules and Silica Particles using Time-Resolved Fluorescence Anisotropy

Abstract

<p> This thesis focuses on the use of time-resolved fluorescence anisotropy (TRF A) for the analysis of peptide-silica and protein-silica interactions. Previous studies from our group have shown that strong ionic binding of the cationic probe rhodamine 6G (R6G) to the anionic surface of silica particles in water provides a convenient labeling procedure to study both particle growth kinetics and surface modification by time-resolved fluorescence anisotropy (TRF A). The decays for R6G dispersed in diluted Ludox silica sols usually fit to a sum of picosecond and nanosecond decay components, along with a significant residual anisotropy component. The first objective of my work was to assess the nature of the R6G:silica interaction to determine the origin of the nanosecond decay component, and ultimately validate the model used to fit the TRFA data and gain further insight into the physical meaning of the anisotropy decay parameters. Our results show the origin of the nanosecond decay component ( ¢2) is due to the presence of a subpopulation of small nanoparticles in the Ludox sol. </p> <p> With the correct physical model in place, we have been able use TRFA ofR6G in aqueous Ludox to monitor peptide adsorption onto the silica particles in situ. Steady-state anisotropy and TRF A of R6G in Ludox sols were measured to characterize the extent of the ionic binding of the probe to silica particles in the presence of varying levels of tripeptides of varying charge, including Lys-Trp-Lys (KWK), N-acetylated Lys-Trp-Lys (Ac-KWK), Glu-Trp-Glu (EWE) and N-acetylated Glu-Trp-Glu (Ac-EWE). R6G showed significant decreases in anisotropy in the presence of cationic peptides, consistent with the addition of cationic peptides blocking the adsorption of the dye to the silica surface. The study shows that the competitive binding method can be used to assess the binding of various biologically relevant compounds onto silica surfaces, and demonstrates the potential of TRF A for probing peptide: silica and protein: silica interactions. </p> <p> We have also extended the application of TRF A to monitor protein adsorption onto plain and modified silica particles using a recently reported cationic long-lifetime quinolinium dye, CG437, which strongly binds to anionic silica particles through electrostatic interactions. In this case, alterations in the rotational correlation time of Ludox particles resulting from increases in the diameter of the rotating body upon binding of protein to the silica surface were monitored. The study shows that TRFA analysis of long-lived cationic probes such as CG437 can provide an effective method to investigate interactions between proteins and modified silica surfaces, extending the utility of the TRF A method. </p>