abstract: 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.
authors.affiliation: Laboratoire de Biologie Physico-Chimique, Faculté des Sciences, 91, Orsay, France
authors.forename: F
authors.initials: F
authors.lastname: Rodier
authors.forename: M
authors.initials: M
authors.lastname: Hill
authors.forename: B
authors.initials: B
authors.lastname: Arrio
authors.forename: C
authors.initials: C
authors.lastname: Parquet
vernacular_title: Un nouveau colorant fluorescent pour la mesure du temps de relaxation brownienne des proteines.