ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 347 (2005) 221–226 www.elsevier.com/locate/yabio
A direct Xuorometric assay for tissue transglutaminase Steve M.F.G. Gillet, Joelle N. Pelletier, JeVrey W. Keillor ¤ Département de Chimie, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montréal, Que., Canada H3C 3J7 Received 23 June 2005 Available onilne 14 October 2005
Abstract Herein we report the design of a direct and continuous Xuorometric assay for determining tissue transglutaminase (TGase) activity. The progress of the TGase-catalyzed reaction of 4-(N-carbobenzoxy-L-phenylalanylamino)-butyric acid coumarin-7-yl ester was monitored as an increase of Xuorescence (exc 330 nm, em 460 nm) due to the release of 7-hydroxycoumarin. Using this assay, we determined the Km of two acceptor substrates, N-acetyl-L-lysine methyl ester and aminoacetonitrile. We also determined the Km of 4-(N-carbobenzoxy-L-phenylalanylamino)-butyric acid coumarin-7-yl ester for its TGase-mediated hydrolysis and for its enzymatic reaction with the acyl acceptor substrates N-acetyl-L-lysine methyl ester and aminoacetonitrile. We ascertained that the Xuorescent substrate was selective toward tissue TGase by testing it with diVerent enzymes, namely microbial transglutaminase (mTGase), Factor XIIIa, papain, and -glutamyl transpeptidase. 4-(N-carbobenzoxyglycinylamino)-butyric acid coumarin-7-yl ester, lacking the benzyl side chain, was also found to be an eYcient Xuorogenic substrate of tissue TGase. Finally, we have shown that this method is applicable to 96-well microtiter plate format. 2005 Elsevier Inc. All rights reserved. Keywords: Enzyme assay; Transglutaminase; Fluorescence; High-throughput screening
Transglutaminases (TGase)1 (amine -glutamyltransferases, EC 2.3.2.13) are a family of Ca2+-dependent enzymes that catalyze acyl transfer reactions. Catalytic activity is exhibited toward the -carboxamide moiety of a protein- or peptide-bound glutamine residue which serves as acyl donor to either protein- or peptide-bound lysine residues or free amines that serve as acyl acceptors, resulting in new intermolecular amidic cross-links [1–3]. Tissue transglutaminase has been identiWed as a contributor to the formation of cataracts and to Celiac disease, *
Corresponding author. Fax: +1 514 343 7586. E-mail address:
[email protected] (J.W. Keillor). 1 Abbreviations used: TGase, transglutaminase; GGT, -glutamyl transpeptidase; Cbz, carbobenzyloxy; DMF, N,N-dimethylformamide; Mops, 3-(N-morpholino)propanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; DIC, diisopropylcarbodiimide; DMAP, 4-dimethylaminopyridine; EtOAc, ethyl acetate; PNPCF, p-nitrophenylchloroformate; BAEE, N-benzoyl-L-arginine ethyl ester; 7-HC, 7-hydroxycoumarin; ZFBC, 4-(Ncarbobenzoxy-L-phenylalanylamino)-butyric acid coumarin-7-yl ester; ZGBC, 4-(N-carbobenzoxyglycinylamino)-butyric acid coumarin-7-yl ester. 0003-2697/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.09.035
and a growing body of evidence suggests that it may be involved in atherosclerosis, inXammation, Wbrosis, diabetes, cancer metastases, autoimmune diseases, and psoriasis (for a review, see [4]). TGase is also suspected to play a role in neurodegenerative diseases (such as Huntington’s disease, Alzheimer disease, Parkinson disease, and supranuclear palsy) associated with an increase in polyglutamine-containing peptides in the brain [5–7]. A few discontinuous methods for monitoring TGase activity have been reported [8,9]. However, in general, continuous methods are more interesting and oVer many practical advantages. In our investigation of the mechanism and inhibition of TGase, we frequently make use of continuous assays involving the formation of a chromophoric anilide product [10], the release of p-nitrophenol [11], or the indirect detection of released ammonia using glutamate dehydrogenase as an auxiliary linking enzyme [12]. A Xuorometric microtiter plate assay based on the measurement of Xuorescence enhancement during the TGase-catalyzed cross-linking of two dansyl derivatives has also been reported [13].
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Fluorometric transglutaminase assay / S.M.F.G. Gillet et al. / Anal. Biochem. 347 (2005) 221–226
was isolated and puriWed as previously published [20]. One unit of GGT was deWned as the amount of enzyme that catalyzes the hydrolysis of 1.0 mol of L--glutamyl-p-nitroanilide per minute at pH 8.0 at 25 °C. Factor XIIIa was purchased from Haematologic Technologies Inc. One unit of Factor XIIIa is equal to its activity in 1 mL of normal plasma. Reagents were obtained from Sigma–Aldrich Chemical Co. Water was puriWed using a Millipore BioCell water puriWcation system. Assay procedure
Enzyme preparation
All assays were performed in triplicate. A standard stock buVer solution was prepared by combining 28 mL of 200 mM Mops buVer (pH 8.0), 20 mL of water, 1.7 mL of 0.1 M CaCl2, and 125 L of 20 mM EDTA. Prior to performing the assays, a “Xuorescence coeYcient” was determined, on a daily basis, by measuring the arbitrary Xuorescence intensities corresponding to Wve concentrations of 7-HC at concentrations ranging from 0 to 10 M in 5% DMF in the stock buVer solution at 25 °C (Fig. 2). Noteworthy is that the values of this coeYcient vary only slightly (