Quantitative nonradioactive CAT assays using fluorescent BODIPY 1-deoxychloramphenicol substrates.

ABSTRACT

We report the development of fluorescent BODIPY 1-deoxychloramphenicol substrates for chloramphenicol acetyltransferase (CAT). These substrates not only simplify and improve quantitation of CAT activity but also extend the linear range of detection. Because the 1-deoxychloramphenicol derivatives have only one acetylation site, the enzyme reaction creates only one product, whereas chloramphenicol and its fluorescent derivatives produce three acetylated products, each of which accumulates at a different rate. Thus, 1-deoxychloramphenicol substrates eliminate the need to account for multiple products and provide a method in which product formation corresponds directly to enzyme activity. These nonradioactive substrates also allow researchers to streamline the standard thin-layer chromatography separation procedure visible results can be obtained within minutes and quantitative results in a few hours. The sensitivity of CAT assays using fluorescent 1-deoxychloramphenicol substrates is comparable to that achieved using [14C]chloramphenicol--between 10(-5) and 10(-6) units of activity in a 1-h reaction--and the linear range extends through 3.6 or more orders of magnitude. We expect that CAT assays employing BODIPY 1-deoxychloramphenicol CAT substrates will be a valuable improvement over other methods currently in use.