Direct and indirect monitoring of peptide-silica interactions using time-resolved fluorescence anisotropy.
The present work extends the application of time-resolved fluorescence anisotropy (TRFA) of a cationic probe rhodamine 6G (R6G) in aqueous Ludox to in situ monitoring of peptide adsorption onto the silica particles. Steady-state anisotropy and TRFA 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). The results were compared to those obtained by direct observation of peptide adsorption using the steady-state anisotropy of the intrinsic tryptophan residue. Ionic binding of the peptides to Ludox particles produced an increase in the steady-state Trp anisotropy that was dependent on the number of cationic groups present, but the limiting anisotropy values were relatively low, indicating significant rotational freedom of the indole residue in the adsorbed peptides. On the other hand, R6G showed significant decreases in anisotropy in the presence of cationic peptides, consistent with the cationic peptides blocking the adsorption of the dye to the silica surface. Thus, R6G is able to indirectly report on the binding of peptides to Ludox particles. It was noteworthy that, while there were similar trends in the data obtained from steady-state anisotropy and TRFA studies of R6G, the use of steady-state anisotropy to assess binding of peptides overestimated the degree of peptide adsorption relative to the value obtained by TRFA. 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 TRFA for probing peptide-silica and protein-silica interactions.
Sui
Jie
J
Department of Chemistry, McMaster University, Hamilton, ON, L8S 4M1, Canada.
Tleugabulova
Dina
D
Brennan
John D
JD
D000327:Adsorption; D005454:Fluorescence Polarization / Q000379:methods; D010455:Peptides / Q000737:chemistry*; D012235:Rhodamines / Q000737:chemistry; D012680:Sensitivity and Specificity; D012822:Silicon Dioxide / Q000737:chemistry*; D013499:Surface Properties; D013997:Time Factors
D010455:Peptides; D012235:Rhodamines; C026188:rhodamine 6G; C024922:Ludox AM; D012822:Silicon Dioxide