Amide bond cleavage monitored continuously through detection of a dansylcadaverine leaving group.
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.
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208.
L
L
Lorand
S N
SN
Murthy
K N
KN
Parameswaran
P T
PT
Velasco
J
J
Wilson
D000577:Amides / Q000378:metabolism*; D000581:Amidohydrolases / Q000032:analysis; D002103:Cadaverine / Q000031:analogs & derivatives* / Q000032:analysis; D002364:Caseins; D007202:Indicators and Reagents; D007700:Kinetics; D013050:Spectrometry, Fluorescence / Q000379:methods; D011503:Transglutaminases / Q000032:analysis / Q000378:metabolism*; D005722:gamma-Glutamylcyclotransferase / Q000032:analysis / Q000378:metabolism*
D000577:Amides; D002364:Caseins; D007202:Indicators and Reagents; D011503:Transglutaminases; D000581:Amidohydrolases; D005722:gamma-Glutamylcyclotransferase; C008542:monodansylcadaverine; D002103:Cadaverine