Figure 1. Spectral properties of EDANS-Dabcyl pair [45] and the flowchart of the experimental design.

(A) EDANS-Dabcyl is a widely used donor-quencher pair. The optimal absorbance and emission wavelengths of EDANS are λ_abs = 336 nm and λ_em = 490 nm respectively, and for Dabcyl, the maximum absorbance wavelength is λ_abs = 472 nm, which, to a large extent, overlap with the emission spectra of EDANS. When they are in a close proximity (10–100 Å), the energy emitted from EDANS will be quenched by Dabcyl, resulting in low or no fluorescence; when they are separated upon substrate cleavage, for example in this design, the fluorescence will increase. Hence from the fluorescence intensity change, the enzyme could be detected continuously and directly. (B) Based on the principle of FRET and our previous study, we chose the optical LC/B cleavage length of VAMP2 (63–85) as the linker between EDANS-Dabcyl.