Ellman’s method

Then purify biotinylated AChE (biot-AChE) on a Biogel A 0.5 m column (1 5 x 90 cm) equilibrated in EIA buffer. The enzymatic profile is established using Ellman s method, and fractions corresponding to the G4 form (major peak) are pooled. Store concentrated biot-AChE at -20°C... 

From 1972 to 1975, Ellman s method (Elhnan et al, 1961), modified for the autoanalyzer, was used. The activity was expressed as ncat/liter. The same method was used for this determination using kinetic methods and instruments, Vitatron (Eefde, Holland) (1976-1990) and Uvicon 952 (Kontron Instruments, Switzerland), from 1991 to 2004. 

The activity of cholinesterases is usually determined by Ellman s method (19). This assay can also be carried out in microtitre plates. Thirty microliters of the ChE dilution and 30 pL of DNTB are added to 210pL of40mM Britton-Robinson-I buffer pH 8.0. The reaction is started by addition of 30 pL of the appropriate acylthiocholine substrate. A microtiter plate reader is used to monitor the development of the absorption at 595 nm. The corresponding slope is used to calculate the esteratic activity. 

S ) is marginal. Therefore, low formation of disulfide bonds is observed. The amount of thiol groups remaining on the polymer can be finally determined by Ellman s method [29]. 

It is known that aryl thiols are more reactive than alkyl thiols due to their lower p a values (5-7) [28]. Aromatic thiols, for example, have already been shown to increase the folding rate of disulfide-cOTitaining proteins more than aUphatic thiols [30, 31]. For a pharmaceutical excipient such as thiolated chitosans, the increased reactivity of the thiol group would further increase its beneficial properties over alkyl thiol chitosan and non-thiolated polymers. Examples of aryl sulfhydryl-bearing compounds covalently attached to chitosan via an amide bond are 6-mercaptonicotinic acid and 4-mercaptobenzoic acid. As mentioned for alkyl thiol chitosan, the amount of free and oxidized thiol groups can be determined by Ellman s method. Examples of developed thiolated chitosans and their degrees of modification are listed in Table 1. 

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. 

For one-step methods, the use of Ellman s reagent (previous section) to yield a similar reversible sulfhydryl blocking group is probably a better choice with protein molecules. 

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... 

Method B Protected Cys(Npys) di-K2K core 39 (4 mg, 8 pmol)1441 was suspended in 0.05 M AcOH (5 mL) and the pH of the mixture adjusted to 8 with 1M NH4OH, and DTT (8 mg) was added. The reaction was spurred by N2 and stirred for 0.5 h. The dark yellow soln was applied to a short Sephadex G10 column and eluted with 0.05 M AcOH. The first eluting fractions, positive by Ellman s test and containing the SH functionalized core, were pooled and Cys(Npys) activated peptide (8.8 pmol, 26 mg) was added. The pH was adjusted to 6 with 1M NaOH. The mixture was purged with N2 and stirred overnight. The liberated 3-nitro-pyridine-2(l//)-thione was eliminated by rapid gel filtration. The MAP was then purified to give 62b by HPLC to eliminate the excess peptide monomer yield 12 mg. 

Probably the most frequently used spectrophotometric method to detect thiol groups, both for non-protein and protein sulphydryl groups involves the use of Ellman s reagent (Scheme 7.5). 5,5 -Dithiobis(2-nitrobenzoic acid), (DTNB) (Ellman 1959) undergoes disulphide exchange with thiol groups and the formation of 5-thio-2-nitrobenzoate anion (TNB) (Scheme 7.6). 

To achieve comparable results (unification of the activity, i.e. one scale), all the activities were recalculated to neat/ liter. Recalculation of the activities was performed simply using U/liter= 16.6667 ncat/hter. However, in the case of different substrates (esters of choline and thiocholine), eorreetion was made according to correlation analysis (y = 2.0188x + 2.2458 and r = 0.9998). Moreover, this correlation was demonstrated previously including not only Ellman s and Winter s methods (Elhnan et al, 1961 Winter, 1960) but also the potentiometric method. 

There are several techniques to evaluate reactivation activity of structurally different AChE reactivators. Probably the most frequent are Eltmann s method (Ellman et al, 1961 Bajgar, 2005) and the potentiostatic method (Kuca and Kassa, 2003). Each one has its advantages and disadvantages. For example, Ellmann s method does not use real substrate ACh, while the potentiostatic method is very time consuming. 

In brief, the method involves the glutathione reductase-catalyzed reduction of GSSG by NADPH, followed by the reaction of GSH with Ellman s reagent, (5,5 -dithiobis-2-nitrobenzoic acid DTNB). The chromophoric product, TNB, has an absorbance between 405 -12 nm. The reaction can therefore be followed spectrophotometrically (Reaction 3). 

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