Dithio-2,2 -bis

The glutathione (GSH) level was assayed by the method of Sedlak and Lindsay (ref. 14), using Ellman s reagent (5,5-dithio-bis-(2-nitrobenzoic acid). Calibration was carried out parallely to each series, using reduced glutathione as a standard. 

Reagents 20 mM Na-K-phosphate buffer pH 7.4, Ellman reagent 5,5"-dithio-bis(p-nitrobenzoic acid) shortly DTNB, or its red analogue 2,2-dithio-... 

Procedure Cholinesterase activity was measured according to the modified biochemical methods developed for crude preparations (Gorunef ah, 1978), using Ellman reagent 5,5"-dithio-bis(p-nitrobenzoic acid) or its red analogue 2,2-dithio-bis-(p-phenyleneazo)-bis-(l-oxy-8-chlorine-3,6) -disulfur acid in the form of sodium salt, which interact with thiocholine salt (Roshchina 2001). Water extracts of vegetative microspores of horsetail (Equisetum arvense) or Hippeastrum hybridum microspores (150 mg of microspores in 30 ml for 1 h) were used. 

The activation of liver FDPase by a variety of sulfhydryl reagents has been examined by Little et al. (46), and their results generally confirm those reported by Pontremoli and his co-workers. In the disulfide exchange reaction 5,5 -dithio-bis(2-nitrobenzoic) acid was most effective (45), and in general, the disulfides were more effective than reagents such as p-mercuribenzoate or iodoacetamide. The nature of the group introduced appears to affect the conformation of the modified enzyme. 

Ellman s reagent, 5,5 -dithio-bis-(2-nitrobenzoic acid), or DTNB, is a compound useful for the quantitative determination of sulfhydryls in solution (Ellman, 1958,1959). The disulfide of Ellman s reagent readily undergoes disulfide exchange with a free sulfhydryl to form a mixed disulfide and release of one molecule of the chromogenic substance 5-sulfido-2-nitrobenzoate, also called 5-thio-2-nitrobenzoic acid (TNB). The intense yellow color produced by the TNB anion can be measured by its absor-... 

The sample to be analysed is diluted in a solution of phosphate buffer, pH8. After stabilisation, a buffered solution of DTNB (5,5 -dithio-bis(2-nitrobenzoic acid) (3,3 -6)) or 3-carboxy-4-nitrophenyl disulphide (otherwise known as Ellman s reagent) is added to the reaction mixture. This reagent is used for specific detection of disulphide bonds and results in a yellow colour which is measured at 405 nm. 

DTNB 5,5 -dithio-bis(2-nitrobenzoic acid) Ellman s Reagent... 

Sulphite and sulphur dioxide can be determined from the changes in colour, produced by their reducing effect on iron(III) in the presence of 1,10-phenanthroline [61,62], TPTZ [63], or ferrozine [64] (for formulae see Ch. 26). Sulphite may also be determined indirectly with Hg(II) chloranilate [59] or thiocyanate [65]. Other reagents for sulphite include 2,6-dichlorophenolindophenol [7] and 5,5 -dithio-bis(2-nitrobenzoic acid) [66-68]. Reaction of SO2 with IOb in the presence of excess of chloride yields ICU", which forms an ion-pair with Pyronine G [69]. 

From Pierce (Rockford, IL) 4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)-toluene (SMPT), /V-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), /V-succinimidyl 5-acetylthioacetate (SATA), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), 2-iminothiolane (2-IT), dithiothreitol (DTT), dimethylformamide (DMF), 5,5 -dithio-bis-(2-nitrobenzoic)acid (DTNB), (Ellman s reagent), 2-mercaptoethanol. 

Figure 8.14 Colourimetric assay. Acetyl Choline Esterase (AChE) assay system involving thio-acetylcholine that is hydrolyzed to thio-choline. This in turn combines with colourless reagent 5,5 -dithio-bis(nitrobenzoic acid) (DTNB) to form yellow coloured 5-thio-2-nitro-benzoic acid (TNB).

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