Several authors have described the NBD-chloride (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) as a fluorogenic reagent of the amino groups and the sulfhydryl groups. It reacts rapidly with the trypsin to produce a stable and highly fluorescent conjugate. The determination of the labelled residues was made with thin layer chromatographies; the lysin residues were preferentially labelled.Fluorescence depolarization measurements of the conjugate were used to determine the brownian relaxation of the enzyme. The temperature dependence of the life-time is very strong and the corrected Perrin's plot demonstrates that the relaxation time of the enzyme decreases rapidly with the temperature.The conclusion is that the NBD-chloride can be used, like the DNS-chloride but is more reactive and stable and its fixation, on trypsin can be followed by measuring the absorbance of the dye at 470 nm.
Rodier
F
F
Laboratoire de Biologie Physico-Chimique, Faculté des Sciences, 91, Orsay, France
Hill
M
M
Arrio
B
B
Parquet
C
C
Un nouveau colorant fluorescent pour la mesure du temps de relaxation brownienne des proteines.