Quantitative nonradioactive CAT assays using fluorescent BODIPY 1-deoxychloramphenicol substrates.
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.
D000107:Acetylation; D001896:Boron Compounds; D002701:Chloramphenicol / Q000031:analogs & derivatives* / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis* / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D005456:Fluorescent Dyes; D007700:Kinetics; D013379:Substrate Specificity
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; C072052:1-deoxychloramphenicol; D002701:Chloramphenicol; D015500:Chloramphenicol O-Acetyltransferase