Measurement using Ellman reagent

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. 

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 demand for routine measurements of cholinc-stcrase activities under field conditions resulted in the development of various field kits. The Test-mate Reid kit, which is currently widely used, is based on the Ellman method. It is a. self-contained portable device comprising a set of reagents and instmetions for use. The Test-mate kit measures AChE and BuChE activities and the hemoglobin content in a drop of blood the activities are automatically nonnalized to 25 C (Wilson, 2001), Oliveira et al (2002) tested three Test-mate kit models (on fetal bovine semm AChE) and compared the results with those measured on a standard spectrophotometer with a 96-wdl microplate reader. The results from the three models showed a discrepancy, and there was also no good agreement between the results of the three models and those obtained from the standard photometric measurement. The authors strongly recommend the use of cholinesterase standards when activities are measured with field kits. 

Acetylthiocholine esters can be used as substrates, the thiocholine formed being measured with chromogenic disulphide reagents, e.g. Ellman s reagent. 

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