Measurement using Ellman

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. 

Monitor the elution of reduced peptide from the column by measuring the absorbance at 280 nm (if peptide absorbs at this wavelength) as well as by performing an Ellman s assay (Section 4.1, this chapter) for sulfhydryl groups using a small aliquot (10-20 pi) of each collected fraction. 

After a carrier protein has been activated with sulfo-SMCC, it is often useful to measure the degree of maleimide incorporation prior to coupling an expensive hapten. Ellman s reagent may be used in an indirect method to assess the level of maleimide activity of sulfo-SMCC-activated proteins and other carriers. First, a sulfhydryl-containing compound such as 2-mercaptoethanol or cysteine is reacted in excess with the activated protein. The amount of unreacted sulfhydryls remaining in solution is then determined using the Ellman s reaction (Chapter 1, Section 4.1). Comparison of the response of the sample to a blank reaction using... 

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. 

The thermal stability of purified Hu BChE in lyophilized (1 mg) or liquid form (10 mg/mL in 50 mM sodium phosphate, pH 8.0+ 10% glycerol + 1 mM EDTA) was examined by measuring enzyme activity following storage at 4°C, 25°C, 37°C, or 45°C. Lyophilized samples were resuspended in 1 mL of 50 mM sodium phosphate buffer, pH 8.0 for measuring BChE activity using the Ellman assay (Ellman et al., 1961). Hu BChE preparations were found to be stable when stored in lyophilized form at 4°C, 25°C, 37°C, or 45°C for over 2 years (Saxena et al., 2005). The enzyme was also stable when stored in liquid form at 4°C and 25°C for 1 year. 

A procedure for identifying certain cholinesterase variants was proposed by Dietz et al. (D15). After a period during which comments and discussion were offered by others working in the field, the method was published in Selected Methods of Clinical Chemistry (D16). This method is based upon the Ellman reaction (ElO), which was used by Ellman et al. (Ell) for the assay of acetylcholinesterase, and by Garry and Routh (G9) for the assay of serum cholinesterase. In these assay procedures, a thiocholine ester is used as the substrate. The thiocholine produced upon hydrolysis reacts with 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB) to yield 5-thio-2-nitrobenzoate anion and other products. The rate of the reaction may be determined by measuring the rate at which... 

Historically, the inhibition of BuChE in plasma has been used to monitor exposure to nerve agents using the classical Ellman colorimetric method (Ellman et al, 1961), or modifications thereof (Woreketal., 1999). This method is used routinely in occupational health monitoring, and could be used to rapidly screen casualties. Its disadvantages are that detection of low-level exposure requires previous baseline measurements, and the assay is non-specific with regard to the inhibitor. 

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