Fluorescence-based assays of lipases, phospholipases, and other lipolytic enzymes.
In choosing an assay, one needs to consider the following questions: What level of sensitivity is required? Must the assay be continuous? Is the substrate readily available; can it be purchased or must it be custom synthesized? How specific is the substrate? How convenient is the method? How compatible is it with monomolecular, micellar, or vesicular substrates? How tolerant is it of added detergents and proteins that may be present? What is the cost of substrates, fluorescent probes, and instrumentation? Of the many methods described in this review, discontinuous assays using natural substrates and derivatization of the products with fluorescent probes are probably the most reliable and most tolerant of reaction conditions. A drawback is the involvement of tedious and time-consuming steps which limit the number of trials that can be performed. Continuous assays, in which changes in fluorescent properties of the probe are monitored, are most convenient for kinetic studies, although they are also most sensitive to reaction conditions and intolerant of added detergents and proteins. One has to carefully consider all of these issues and choose a method best suited to the enzyme, the particular information one wants to obtain, and the availability of substrates, probes, and instrumentation. Hopefully, increased commercial availability of fluorescent substrates and probes will make these choices easier. Nevertheless, the search goes on for better, more sensitive and convenient fluorescent assays.
Hendrickson
H S
HS
Department of Chemistry, St. Olaf College, Northfield, Minnesota 55057.
D000818:Animals; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D006801:Humans; D007202:Indicators and Reagents; D008049:Lipase / Q000032:analysis*; D008066:Lipolysis; D010740:Phospholipases / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D010740:Phospholipases; D008049:Lipase