In addition to fusion proteins, ubiquitin derivatives conjugated with a fluorophore have been reported as substrates for biochemical DUB assays. A frequently used coumarin-based substrate is ubiquitin7-amido-4-methylcoumarin (Ub-AMC). DUBs catalyze the release of the AMC moiety, which is directly attached to the C-terminus of ubiquitin, and liberation of the fluorophore results in de-quenching of the fluorescent signal (Hassiepen et al., 2007). The excitation/emission range of this fluorophore is 380nm/460nm respectively. The use of this substrate for determining steady-state kinetic parameters in a number of DUB assays was first described by Dang et al. (1998).
Dang LC, Melandri FD and Stein RL (1998) Kinetic and mechanistic studies on the hydrolysis of ubiquitin C-terminal 7-amido-4methylcoumarin by deubiquitinating enzymes. Biochemistry 37, 1868-1879.
Hassiepen U, Eidhoff U, Meder G, Bulber JF, Hein A, Bodendorf U, et al. (2007) A sensitive fluorescence intensity assay for deubiquitinating proteases using ubiquitin-rhodamine110-glycine as substrate. Anal Biochem 371, 201-207.