A Direct Continuous Spectrophotometric Assay for Transglutaminase Activity

Abstract

Herein we report the development of a direct and continuous spectrophotometric method for determining transglutaminase (TGase) activity by using N,N-dimethyl-1,4-phenylenediamine (DMPDA) as a γ-glutamyl acceptor substrate and carbobenzyloxy-l-glutamylglycine (Z-Gln-Gly) as a typical peptide γ-glutamyl donor substrate. The transamidation activity of TGase can thus be followed by monitoring the increase of absorbance of the resulting anilide product at 278 nm. The extinction coefficient of the authentic, independently synthesized anilide was determined to be ϵ = 8940 ± 55 M−1 cm−1. Using this assay, we determined the apparent KM of DMPDA to be 0.25 mM, which compares favorably to the apparent KM values determined for other acceptor substrates under conditions where Z-Gln-Gly is also used as the donor substrate, such as N-acetyl-l-lysine methyl ester (9.6 mM) and methylamine (13.1 mM). Finally, the sensitivity of this assay technique was established through the measurement of irreversible inhibition constants for iodoacetamide, determined to be KI = 75 ± 11 nM and kinact = (120 ± 1) × 105 M−1 min−1.