Ellmans reagent

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- 

Mix the protein solution with an equal volume of the Ellman s reagent solution and react for 15 min at room temperature. 

Purify the modified protein from excess Ellman s reagent and reaction byproducts by dialysis or gel filtration. A measurement of sulfhydryl content may be done by reading the absorbance of the modification reaction at 412 nm (e = 1.36 X 104M icm i)versusaseries of sulfhydryl standards treated in the same manner (e.g., cysteine). 

To deblock the TNB-modified sulfhydryl residues, treat the protein with an excess of DTT according to the protocol described in section 1.1.4.1, DTT. 

Dissolve the protein to be blocked at a concentration of l-10mg/ml in 0.1 M sodium phosphate, pH 8.0. 

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Fig. 10 Histochemical staining of Hippeastrum hybridum pollen ( A), Epiphyllium hybridum pollen (B) and part of pistil of Hippeastrum hybridum (C) by red analogue of Ellman reagent. Blue color means the presence of cholinesterase.

Experiment 2. Cholinesterase as a sensor on the cell surface A target of the allelochemical may also be a surface sensor-cholinesterase (Fig. 10). If after the staining with Red analogue of Ellman reagent the blue colour is absent in the allelochemical treated microspore, possible target is the enzyme (Roshchina, 2001a,b) as for alkaloid berberine tested. If after the treatment by the test allelochemical, the colour is absent or light, the compound inhibits the enzyme (also see biochemical assay in Chapter 11). 

Principle The determination of acetylthiocholine hydrolysis, based on the absorbance at 412 nm of the yellow product - complex of thiocholine with Ellman reagent. 

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. 

FIGURE 58.1. Acetylthiocholine is hydrolyzed by AChE to acetic acid and thiochohne, which forms yellow 5-mercapto-2-nitrobenzoic acid after reaction with Ellman reagent. 

Reagents for oxidation 5,5 -dithio-hw-(2-nitrobenzoic acid) (DTNB, Ellman reagent)", silver triflate (AgOTf)", thallium trifluoroacetate (Tl(tfa)3), iodine, and guanidine HCT (Fluka or Sigma-Aldrich). 

J8A0. Titration of thiols with 5 -dithiobis( 2-nitrobenzoate) (DTNB-, Ellmans reagent)... 

Eyer P, Worek F, Kiderlen D, Sinko G, Stuglin A, Simeon-Rudolf V, Reiner E. Molar absorption coefficients for the reduced Ellman reagent reassessment. Anal Biochem, 2003 312 224—227. 

The Fab fraction is eluted in the void volume, concentrated to 5 mg/mL, and treated with a 1/100 volume of DTNB (Ellman reagent) dissolved in DMF (80 mg/mL). 

Ellman reagent 1415 Elspar 537 ELUTES trial 315 Embryoid body 286 Embryonic antigen 288 Embryonic development 287 Embryonic germ (EG) 278 Embryonic renal progenitor 357... 

Eyer. P Worek, F Kiderlen, D., Sinko, G., Stuglin. A., Simeon-Rudolf, V., and Reiner, E. (2003). Molar absorption coefficients for the reduced Ellman reagent Reassessment. Anal. Biochem. 312,224-227. 

Example 2 Kinetic analysis of the reaction of Ellmans reagent DTNB) and thioglycerol RS. 

The classic reagent is 5,5 -dithiobis-(2-nitrobenzoic acid) or DTNB, sometimes known as Ellmans reagent (32), which is absolutely specific for the thiol side... 

Dithiobis[2-nitrobenzoic acid. Ellmans reagent. DTNB... 

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