Amide bond cleavage monitored continuously through detection of a dansylcadaverine leaving group

• Published in: Biochemical and biophysical research communications Vol. 186; no. 1; p. 334
• Main Authors: Lorand, L, Murthy, S N, Parameswaran, K N, Velasco, P T, Wilson, J
• Format: Journal Article
• Language: English
• Published: United States 15-07-1992
• Subjects: Amides - metabolism; Amidohydrolases - analysis; Cadaverine - analogs & derivatives; Cadaverine - analysis; Caseins; gamma-Glutamylcyclotransferase - analysis; gamma-Glutamylcyclotransferase - metabolism; Indicators and Reagents; Kinetics; Spectrometry, Fluorescence - methods; Transglutaminases - analysis; Transglutaminases - metabolism
• ISSN: 0006-291X
• DOI: 10.1016/S0006-291X(05)80812-5

The transglutaminase-catalyzed incorporation of the fluorescent amine, dansylcadaverine, into casein derivatives, such as N,N-dimethylcasein, is accompanied by a large increase in intensity of emission (Lorand et al., Anal. Biochem. 44, 221-231, 1971). We have sought to make use of this sensitive detection device for the continuous, on-line monitoring of an amide-splitting reaction in which dansylcadaverine served as the leaving group. The transglutaminase-coupled test system comprised gamma-glutamyldansylcadaverine as the first substrate and gamma-glutamylamine cyclotransferase as the enzyme responsible for releasing dansylcadaverine from the gamma-amide. At close to saturating levels of transglutaminase, the measured rate of increase of fluorescence, i.e. the steady-state rate of dansylcadaverine incorporation into N,N-dimethylcasein, showed a near-linear relationship with the concentration of gamma-glutamylamine cyclotransferase present in the assay mixture. The general approach developed may be applicable to the assay of other amide cleaving enzymes.