Abstract: Activity of enzymes and enzyme conjugates is detected using novel substrates made from a class of fluorophores, generally including quinazolinones (quinazolones), benzimidazoles, benzothiazoles, benzoxazoles, quinolines, indolines, and phenanthridines, having the general formula: ##STR1## where carbon atoms of --C.sup.1 .dbd.C.sup.2 -- are joined to complete a first 5- or 6-membered aromatic ring which optionally contains one of the hetero atoms N, O or S,and carbon atoms of --C.sup.4 --N.dbd.C.sup.3 -- are joined to complete a second 5- or 6-membered aromatic ring that contains a nitrogen between C.sup.3 and C.sup.4 and optionally contains an additional hetero atom N, O or S,where the first and second aromatic rings may be joined by a 5- or 6-membered bridging ring that contains at least the C.sup.2 from the first aromatic ring and the C.sup.

Abstract: Fluorescent compounds useful in the determination of chloramphenicol acetyltransferase (CAT) enzyme activity are described. The compounds are fluorescent derivatives related in structure to chloramphenicol comprising a base, ##STR1## substituted at one to five aromatic ring positions by substituents, which may be the same or different, that are alkyl-, hydroxy-, alkoxy-, aryl-, halo-, nitro-, amino-, alkylamido-, or arylamido-; and a fluorescent moiety linked to the base at --NH-- by an aliphatic chain of 1-12 atoms other than hydrogen, where such atoms are carbon, nitrogen, oxygen, or sulfur, or combinations thereof. The substrate compounds are acylated in the presence of CAT to produce fluorescent mono- and diacylated products, which are then physically separated from the reaction mixture and quantitated by means of their fluorescence and/or absorbance.