ANALYTICAL BIOCHEMISTRY 161, 64 -69 (1987) Quantitation of Aspartate Aminotransferase Isoenzymes after Eiectrophoretic Separation DANIEL A. NEALON AND ROBERT REJ Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201 Received July 16, 1986 A scheme for the quantitative detection of aspartate aminotransferase isoenzymes and multi- ple forms after electrophoretic separation is described. Glutamate generated from the amino- transferase reaction is quantitated by using the glutamate dehydrogenase/diaphorase-coupled enzyme system to form a formazan dye. Product inhibition of aspartate aminotransferase by oxaloacetate is prevented by including oxaloacetate decarboxylase in the overlay reagent. Re- sults compare favorably with those of an immunochemical precipitation procedure. The method can also be used to detect quantitatively subforms and atypical forms (genetic variants, unoglobulin-enzyme complexes) of aspartate aminotransferase. © 1987 Academic Press, Inc. KEY WORDS : aspartate aminotransferase ; aminotransferases ; electrophoresis ; gel staining. Mammalian aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransfer- ase, EC 2.6.1.1, AspAT)1 exists in two pre- dominant isoenzymic forms: an anodic or soluble (s-AspAT) form and a cathodic or mitochondria) (m-AspAT) form. These have sufficiently different isoelectric points so that they can be easily separated by electrophore- sis, and virtually all common electrophoretic media have been applied to separate them (1). A number of procedures have been used to detect AspAT isoenzyme activity after elec- trophoretic separation. Some visualization procedures suffer from the difficulty of quantitating a lack of fluorescence on a fluo- rescent background or a lack of color against a highly colored background (1). Staining with an azoene dye such as fast violet B is not specific and many of these dyes can also react with 2-oxoglutarate (2,3). Additional prob- Abbreviations used : AspAT, aspartate aminotrans- ferase ; s-AspAT, anodic or soluble AspAT; m-AspAT, cathodic or mitochondrial AspAT; INT, iodonitrotetra- zolium violet. lems with these procedures include product inhibition of the enzyme during the reaction (4) and diffusion and decarboxylation of ox- aloacetate (5). Cysteine sulfinate reactivity of AspAT to form S®and subsequent reduction of tet- razolium dyes have been used to detect the Isoenzymes after electrophoresis (6,7). How- ever, the ratio of activity between m- and s-AspAT for this substrate and aspartate differs by about fivefold (8), making absolute quantitation of isoenzyme activity difficult. Visualization techniques using glutamate de- hydrogenase (EC 1.4.1.3) are also usually semiquantitative, as oxaloacetate accumu- lates during the enzyme reaction and inhibits AspAT activity, particularly that of m- AspAT (4). In this report we describe a sensitive quan- titative procedure based on a glutamate de- hydrogenase- and diaphorase-coupled en- zyme system (9) in which oxaloacetate decar- boxylase (EC 4.1.1.3) is used to remove oxaloacetate. The procedure was evaluated by using purified human Isoenzymes and tis- sue extracts from various species. 0003-2697/87 $3.00 64 Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.