A direct method to visualise the aryl acylamidase activity on cholinesterases in polyacrylamide gels

The o-nitroaniline liberated from o-nitroacetanilide by the action of aryl acylamidase activity on cholinesterases, in the presence of nitrous acid formed a diazonium compound. This compound gave an azo dye complex with N-(1-napthyl)-ethylenediamine, which appeared as purple bands in polyacrylamide gels. Treating the stained gels with trichloroacetic acid followed by Tris-HCl buffer helped in fixation of the stain in the gels. By using specific inhibitors for acetylcholinesterase and butyrylcholinesterase, respectively, differential staining for the aryl acylamidase activities on butyrylcholinesterase and acetylcholinesterase in a sample containing both these enzymes has been demonstrated. A linear relationship between the intensity of colour developed and activity of the enzyme was obtained.A novel method to visualise the aryl acylamidase activity on cholinesterases in polyacrylamide gels has been developed.Cholinesterases (ChEs) are evolutionarily conserved type B carboxylesterase enzymes that share extensive sequence homology. In vertebrates two types of ChEs were identified based on their distinct substrate specificity and inhibitor sensitivity. The acetylcholinesterase (AChE; EC 3.1.1.7) specifically catalyses the hydrolysis of acetylcholine and is subjected to marked inhibition by its own natural substrate. In contrast, butyrylcholinesterase (BChE; EC 3.1.1.8) is capable of degrading a wider range of choline esters and is not inhibited by its substrate [1, 2]. AChE is selectively inhibited by BW 284c51, while BChE is specifically inhibited by tetraisopropylpyrophosphoramide (iso-OMPA) [3]. AChE is widely distributed in the nervous system and its role in rapidly terminating nerve impulse by hydrolysing acetylcholine in cholinergic synapses is well documented [1]. BChE is produced in the liver and enriched in the circulation. In addition, it is also present in adipose tissue, intestine, smooth muscle cells, white matter of the brain and many other tissues [4]. T