Fluorescence Enhancement of Fluorescent Unnatural Streptavidin by Binding of a Biotin Analogue with Spacer Tail and Its Application to Biotin Sensing
(a) Fluorescence spectra of the Trp120BFLAF mutant streptavidin in the presence of the pairs of 0 nM (–◊–), 20 nM (–□–), 50 nM (––), 80 nM (–×–), or 100 nM (–+–) of natural biotin and 100 nM of biotin-(AC5)2-hydrazide. Marked fluorescence enhancement was observed for the Trp120BFLAF mutant streptavidin by addition of biotin-(AC5)2-hydrazide. Decrease of fluorescence of Trp120BFLAF mutant streptavidin was observed upon competitive biotin addition in the presence of 100 nM of biotin-(AC5)2-hydrazide. (b) The fluorescence intensities of the Trp120BFLAF mutant streptavidins in the presence of the pairs of various concentrations of natural biotin and 100 nM of biotin-(AC5)2-hydrazide at 514 nm were plotted. fluorescence intensity (au) of the Trp120BFLAF mutant streptavidin depended on the concentration of free biotin. All of the fluorescence spectra of mutant streptavidins were excited at 490 nm.