Archives of Biochemistry and Biophysics 440 (2005) 118–122 www.elsevier.com/locate/yabbi 0003-9861/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2005.06.003 Inhibition of human plasma cholinesterase by malachite green and related triarylmethane dyes: Mechanistic implications Tuba KüçükkÂlÂnç, Ãnci Özer ¤ Department of Biochemistry, School of Pharmacy, Hacettepe University, 06100 Ankara, Turkey Received 15 April 2005, and in revised form 7 June 2005 Available onlilne 1 July 2005 Abstract The inhibitory eVects of the cationic triarylmethane (TAM + ) dyes, pararosaniline (PR + ), malachite green (MG + ), and methyl green (MeG + ) on human plasma cholinesterase (BChE) were studied at 25 °C in 100 mM Mops, pH 8.0, with butyrylthiocholine as substrate. PR + and MG + caused linear mixed inhibition of enzyme activity. The respective inhibitory parameters were K i D 1.9 § 0.23 M,  D 13 § 48,  D 0 and K i D 0.28 § 0.037 M,  D 23 § 7.4,  D 0. MeG + acted as a competitive inhibitor with K i D 0.12 § 0.017 M (, 1, , not applicable). The K i values were within the same range reported for a number of ChE inhibitors including propidium ion, donepezil, and the phenothiazines, suggesting that TAM + s are active site ligands. On the other hand, the  values failed to correlate with values previously reported for a number of ChE inhibitors. It appears that mixed inhibition is the com- bined result of more than one type of binding and S–I interference. The impact of ligands at the choline-speciWc and peripheral anionic sites (or, possibly, accessory structural domains) on BChE activity needs to be studied in further detail.  2005 Elsevier Inc. All rights reserved. Keywords: Plasma cholinesterase; Enzyme inhibition; Pararosaniline; Malachite green; Methyl green; Triarylmethane dyes Acetylcholinesterase (AChE, EC 3.1.1.7) and butyr- ylcholinesterase (BChE, 1 EC 3.1.1.8), encoded by distinct genes, are kin enzymes that are similar in overall struc- ture and catalytic mechanism [1–5]. Both enzymes have been studied extensively [6–10], owing to the critical role of AChE in cholinergic neurotransmission and to the apparent involvement of BChE in the metabolism/ detoxiWcation of carboxylic and phosphoric acid esters, a role suggested by the high levels of the enzyme in liver, lung, and plasma [1,11–13]. A further point of interest, disclosed by recent studies, is that the two cholinester- ases may have a coordinated impact on a number of developmental processes and overall brain function, as well as on the progression of Alzheimer’s disease [14–18]. This information has added new dimensions to research on AChE and BChE, including an intensiWed search for cholinesterase inhibitors as therapeutic agents [19–21]. The following is a report on the eVect of malachite green and related cationic triarylmethane dyes on the activity of human plasma cholinesterase. TAM + dyes (Fig. 1) have been traditionally used as industrial colo- rants and as disinfectants in the Wsh farming industry and medicine [22–24]. The biocidal eVects have generally been attributed to the capacity of the dyes to intercalate nucleic acids and to induction of radical-mediated redox changes [25,26]. Earlier studies in this laboratory showed that TAM + s can form adducts with a variety of proteins and protein-related nucleophiles, pointing to a further mode of TAM + action by modulation of enzyme activity [27]. The speciWcity of cholinesterases for cationic * Corresponding author. Fax: +90 312 3114777. E-mail address: iozer@hacettepe.edu.tr (I . Özer). 1 Abbreviations used: BChE, butyrylcholinesterase (plasma cholines- terase); BTC, butyrylthiocholine; DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid); MeG + , methyl green; MG + , malachite green; Mops, 3-(N-mor- pholino)propanesulfonic acid; PR + , pararosaniline; TAM + , triarylmethane.