ULTRAMICRO ANALYSIS OF ENZYMES AND SUBSTRATES BY ENZYMATIC AMPLIFICATION REACTIONS “ENZYMATIC CYCLING”

Abstract The principle and kinetics of enzymatic cycling are described. The enzymatic cycling reaction is catalyzed by a couple of enzymes and amplifies stoichiometrically a pair of substrates participating in the reaction. Many biological substances can be converted quantitatively to substrates that can be amplified and measured by cycling reactions. Three cyclings in routine use (NAD, NADP and CoA cyclings) are summarized and compared. Each cycling provides an amplification of more than 10,000 times per hour and the combination of these cyclings (double and triple cyclings) results in an even larger gain in sensitivity. Theoretically single molecules can be measured by using the triple cycling. As examples, the determination of ATP and acetylcholine synthesizing enzyme (choline acetyltransferase, EC 2.3.1.6) are described. A novel method ( popcorn ball technique ) discriminating CoASH and acetyl-CoA, which are the substrates amplified by CoA cycling, is explained in detail. The cycling reactions are believed to be capable of replacing many radiometric methods.