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.
Lorand
L
L
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208.
Murthy
S N
SN
Parameswaran
K N
KN
Velasco
P T
PT
Wilson
J
J
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