Fluorescence Enhancement of Fluorescent Unnatural Streptavidin by Binding of a Biotin Analogue with Spacer Tail and Its Application to Biotin Sensing
(a) Fluorescence spectrum of the Tyr83BFLAF mutant streptavidin only (–◯–); fluorescence spectrum of the Tyr83BFLAF mutant streptavidin in the presence of 100 nM of natural biotin (––) or 100 nM of biotin-(AC5)2-hydrazide (–□–); there was no change in the fluorescence intensity (au) upon addition of natural biotin or biotin-(AC5)2-hydrazide for Tyr83BFLAF mutant streptavidin. (b) Fluorescence spectra of the Trp120BFLAF mutant streptavidin only (–◊–), in the presence of 100 nM of biotin (–■–), 20 nM (–□–), 50 nM (––), 80 nM (–×–), 100 nM (––), 200 nM (–◯–), or 500 nM (–+–) of biotin-(AC5)2-hydrazide. Marked fluorescence enhancement was observed for the Trp120BFLAF mutant streptavidin by addition of biotin-(AC5)2-hydrazide. (c) Dependence of the fluorescence enhancement of Trp120BFLAF mutant streptavidin on the concentration of added biotin-(AC5)2-hydrazide (20 nM to 100 nM) was observed. The fluorescence intensities of the Trp120BFLAF mutant streptavidins in the presence of various concentration of biotin-(AC5)2-hydrazide at 514 nm were plotted. All of the fluorescence spectra of mutant streptavidins were excited at 490 nm.