2-Aminoanthracene as an Analytical Tool with the Acetylation Reaction Catalyzed by Arylamine N-Acetyltransferase L. Servillo, 1 C. Balestrieri, M. Boccellino, M. L. Balestrieri, L. Quagliuolo, and A. Giovane Department of Biochemistry and Biophysics, 2nd University of Naples, Via Costantinopoli 16, 80131 Naples, Italy Received March 18, 1999 The polynuclear aromatic amine, 2-aminoanthra- cene, was found to be acetylated with high efficiency in the presence of acetyl-CoA by pigeon liver arylamine N-acetyltransferase (EC 2.3.1.5). As a conse- quence of acetylation the fluorescence properties of the compound dramatically change and the reaction time course can be easily followed fluorometrically at the emission wavelength of 425 nm upon excitation at 360 nm. When 2-aminoanthracene is employed with pigeon arylamine N-acetyltransferase, as the ultimate acceptor of the acetyl group in coupled fluorometric assays, it is possible to measure enzymatic activities, such as pyruvate dehydrogenase or carnitine acetyl- transferase, in continuous assays rapidly and with high sensitivity or to determine with as much sensi- tivity important metabolites such as acetylcarnitine or acetyl-CoA. © 1999 Academic Press Key Words: arylamine N-acetyltransferase; pyruvate dehydrogenase; carnitine acetyltransferase; acetyl- carnitine; acetyl-CoA. It is well known that aromatic amines in several species including humans undergo an effective bio- transformation through an N-acetylation step (1) cat- alyzed by the enzyme acetyl-CoA:arylamine N-acetyl- transferase (NAT) 2 (EC 2.3.1.5). This polymorphic enzyme shows a broad specificity (2) and can acetylate more or less rapidly a wide class of aromatic monocyclic and polycyclic amines by utilizing acetyl-CoA accord- ing to the reaction: Aryl-NH 2 + CH 3 COSCoA 3 Aryl-NHCOCH 3 + CoASH. Since as a consequence of acetylation, owing to the electron-withdrawing character of the acetyl moiety, in many cases the arylamine spectral properties consid- erably change, the acetylation reaction can be easily followed spectrophotometrically from the absorbance changes that the acetylated arylamine undergoes with respect to the parent arylamine. Therefore, NAT was used in coupled spectrophotometric assays to deter- mine enzymatic activities producing acetyl-CoA by uti- lizing, as in the case of pyruvate dehydrogenase, vari- ous arylamines, such as p-(p-aminophenylazo)-benzene sulfonic acid (3) or p-nitroaniline (4), as the ultimate acceptors of the acetyl group. In one case a coupled fluorometric assay was proposed which was based on the fluorescence variations of a dye upon acetylation (5). The enzyme from pigeon liver has often been used in those assays as an enzymatic source for NAT be- cause of its relative abundance in this species, al- though, more recently, a method for a convenient prep- aration of the enzyme from chicken liver has been reported (6). To study the NAT activity in extracts of cultured cells, we tested the capacity of a variety of polycyclic aromatic amines, some of which with good fluorescence properties, to be acetylated. In the present paper, we report that 2-aminoanthracene has excellent charac- teristics, in terms of fluorescence properties and en- zyme affinity, to be utilized to measure NAT activity at very low levels. Moreover, when 2-aminoanthracene is employed with pigeon liver NAT as the ultimate accep- tor of acetyl group in coupled fluorometric assays, it is possible to measure enzymatic activities, such as pyru- vate dehydrogenase or carnitine acetyltransferase, in continuous coupled assays rapidly and with high sen- 1 To whom correspondence should be addressed. Fax: +39-815665863. E-mail: luservil@unina.it. 2 Abbreviation used: NAT, arylamine N-acetyltransferase. 0003-2697/99 $30.00 105 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. Analytical Biochemistry 273, 105–110 (1999) Article ID abio.1999.4200, available online at http://www.idealibrary.com on