Enzymatic Activity Revealed Fluorescence methods are also being used quite elegantly for enzymatic studies. Enzymes can be exposed to substrates that change their intrinsic fluorescence when processed by the target protein. Typically, the substrate is nonfluorescent and converted to a fluorophore through one or more reactions. These kits combine peroxidating secondary reactions and can detect glucose, monoamine and glutamate oxidases, acetylcholinesterases, PC-specific phospholipase C, phospholipase D, and sphingomyelinase. A standard protocol is to label a protein with many copies of a fluorophore.
Proteolytic activity is measured by release of small fluorescent peptides.
Another way to study cleavage of a substrate is to combine a quencher and a fluorophore on the same molecule. Rupture of intramolecular bonds separates the fluorophore and quencher and leads to emission of fluorescence. This general scheme can also be applied to enzymes that cleave oligonucleotides. As the fluorophore is released, it becomes highly fluorescent. The activity of protein kinases can also be studied by fluorescence methods.