A direct continuous spectrophotometric assay for transglutaminase activity

Herein we report the development of a direct and continuous spectrophotomet ric method for determining transglutaminase (TGase) activity by using N,N-d imethyl-1,4-phenylenediamine (DMPDA) as a gamma-glutamyl acceptor substrate and carbobenzyloxy-L-glutamylglycine (Z-Gln-Gly) as a typical peptide gamm a-glutamyl donor substrate. The transamidation activity of TGase can thus b e followed by monitoring the increase of absorbance of the resulting anilid e product at 278 nm. The extinction coefficient of the authentic, independe ntly synthesized anilide was determined to be epsilon = 8940 +/- 55 M-1 cm( -1). Using this assay, we determined the apparent K-M of DMPDA to be 0.25 m M, which compares favorably to the apparent K-M values determined for other acceptor substrates under conditions where Z-Gln-Gly is also used as the d onor substrate, such as N-acetyl-L-lysine methyl ester (9.6 mM) and methyla mine (13.1 mM), Finally, the sensitivity of this assay technique was establ ished through the measurement of irreversible inhibition constants for iodo acetamide, determined to be K-I = 75 +/- 11 nM and k(inact) = (120 +/- 1) x 10(5) M-1 min(-1). (C) 2000 Academic Press.