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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... [Pg.62]

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. [Pg.882]

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. [Pg.9]

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]. [Pg.97]

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. [Pg.98]

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. [Pg.390]

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. [Pg.166]

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... [Pg.768]

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. [Pg.150]

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). [Pg.228]

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. [Pg.882]

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. [Pg.1009]

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). [Pg.84]


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See also in sourсe #XX -- [ Pg.828 , Pg.878 ]




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Ellman method

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