Recent advances in enzyme assays.

Enzyme assays for high-throughput screening and enzyme engineering, which are often based on derivatives of coumarin, nitrophenol, fluorescein, nitrobenzofurazane or rhodamine dyes, can be divided into two categories: those that depend on labelled substrates, and those that depend on sensing the reactions of unmodified substrates. Labelled substrates include, for example, fluorogenic and chromogenic substrates that generate a reporter molecule by beta-elimination, fluorescence resonance energy transfer (FRET) substrates and isotopic labels for enantioselectivity screening. By contrast, endpoint sensing can be done using amine reagents, fluorescent affinity labels for phosphorylated proteins, or synthetic multifunctional pores. Sensing assays can also be done in real time by using, for example, aldehyde trapping to follow vinyl ester acylation in organic solvent or calcein-copper fluorescence for sensing amino acids. The current trend is to assemble many such assays in parallel for enzyme profiling and enzyme fingerprinting.