Ellman procedure

Gorun, V., Proinov, I., Baltescu, V., Balaban, G. and Barzu, O. (1978). Modified Ellman procedure for assay of cholinesterases in crude enzymatic preparations. Analytical Biochemistry 86 324-326. 

Test-Mate Assay In addition to the laboratory-based methodologies, a field deployable unit is commercially available, the Test-mate ChE system (EQM Research Inc., Cincinnati, OH). This method is also based on the Ellman procedure and is supplied as a kit containing reagents to separately measure erythrocyte AChE and plasma-BChE with a battery-operated, photometric analyzer (Taylor et al., 2003). A specific serum BChE inhibitor (As 1397, or 10-(oi-diethylaminopropionyl)-phenothia-zone) is included in the kit and is required to measure AChE (after a period of incubation). Two capillary... 

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. 

Reduced Sulfur Compounds in Marine Sediments. To determine the applicability of the bimane-HPLC technique to measure reduced sulfur compounds in sediment porewater samples, we compared the results of the methylene blue method of Cline (26). the DTNB procedure of Ellman (28) and the bimane-HPLC procedure outlined above. Cores included came from a Spartina foliosa marsh in Mission Bay (near San Diego, California), and an evaporation pond for the production of salt in south San Diego Bay (Table I). 

Compound 17, the final product resulting from stealth inhibitor 14, was subsequently synthesized and its inhibitory potency evaluated using the spectrophoto-metric procedure of Ellman et al. (Fig. 4) [13]. Mixed inhibition of compound 17 was recorded, with a competitive inhibition constant (Kt) of 47 nM, and a noncompetitive constant (ocAT ) of 103 nM, while the Michaelis constant for ATCh was determined to be 114 pM. 

Schmidt and coworkers [80] developed the pentafluorophenyl ester method for synthesizing cyclopeptides, particularly applicable for 13- and 14-membered ansa peptides. This procedure is superior to the p-nitrophenyl ester method in respect to short reaction time and easy separation of products. Evans and Ellman [81] applied this method to the synthesis of the cyclic tripeptide K-13 146). As shown in Scheme 48, the reaction of the linear precursor 143 with pentafluorophenol and DCC afforded pentafluorophenyl ester 144 in 87-93% yield. Then, under catalytic hydrogenation condition in the presence of a mild base and ethanol, 144 was cyclized to 145 in up to 70% yield. There are more applications of the pentafluorophenyl ester procedure in recent literature [82]. 

Following its introduction in 1959 (Ellman), this sensitive spectro-photometric procedure for the determination of thiols has gained wide acceptance. The mixed disulfide product can be exploited as an intermediate in the preparation of other useful derivatives of the cysteinyl residue. 

Methods for the detection of cholinesterase activity can generally be divided into four groups, electrometric, colorimetric,titrimetric and tintometric methods, with the last being the one mainly used in the field. The colorimetric method developed by Ellman et al. is the most frequently used procedure. Titrimetry is extremely accurate and precise, but rarely used because of its high cost and complexity. Electrometric methods are less sensitive than colorimetric methods and less accurate than titrimetric methods. 

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... 

Sulfhydryl content can be determined directly by using any thiol assay, typically by using Ellman s reagent, although other more sensitive procedures exist (47), or indirectly by release of some reporter molecule on deprotection of crosslinkers. For example, many reports use the absorbance of 2-thiopyridone at 343 nm after treatment of pyridyldithio-modified proteins with DTT. Extreme caution should be used when interpreting results from these indirect methods since in our experience treatment of antibodies with DTT, even under mild conditions, results in subtle changes in the baseline absorbance at 343 nm and consequent erroneous estimates of thiol content. 

In a second report, the Bergman and Ellman groups improved on their original procedure for the arylation of heterocycles with aryl halides [103]. The key improvements over the previous method were three-fold (1) the use of the hindered tertiary amine base Pr.NBu in replacement of Et3N (2) microwave irradiation for rapid (<1 h), high temperature (250 °C) reactions in 1,2-dichlorobenzene (3) the use of bulky bicyclic trialkylphosphine ligands of the phobane family in replacement of PCy3 (Scheme 15). Under typical reaction conditions, the heterocycle... 

Patterson, A.W, Wood, W.J.L., and Ellman, J.A. (2007) Substrate activity screening (SAS) a general procedure for the preparation and screening of a fragment-based non-peptidic protease substrate library for inhibitor discovery. Nature Protocols, 2 (2), 424—433. 

Assays of residual AChE were made by the Ellman spectrophotometric procedure based on acetylthiocholine and the cleavable indicator, 5,5 -dithiobis-2-nitrobenzoic acid (17). The negative logarithm of the molar concentration causing 50% inhibition, pI50, is derived from the inhibition-concentration plot. 

The standard methodology for determining blood ChE inhibition is based on the measurement of the enzymatic products derived from either acetylcholine or acetylthiocholine as substrates. The simplest and most convenient method is a colorimetric procedure (Ellman et al., 1961) in... 

Besides the applications of pulse combustors as residential, space heater and propulsion procedure, etc., a more active area is that of drying. Ellman et al. (1966) first carried out experimental studies of lignite drying using a 205 kW pulse... 

A diastereoselective approach to these chiral building blocks was developed by Ellman (Scheme 8.15) [40]. Thus, N-tert-butylsulfinyl aldimines 57 were employed as electrophiles in the rhodium/phosphine 58-catalyzed addition of arylboronic acids. Whereas Ellman s procedure requires heating, Batey discovered that an amine base allows the reaction to take place at ambient temperature [41]. However, it is worth noting that Ellman s process can also be carried out in an enantioselec-tive way by using N-diphenylphosphinoyl aldimines 48 as substrate and Degu-PHOS (56) as ligand (87-97% yield, 88-94% ee) (Scheme 8.14 Figure 8.4). 

Ellman, Bergman, and coworker reported a rhodium-catalyzed procedure for the synthesis of pyridines from alkynes and a,/ -unsaturated N-benzyl aldimines and ketimines in 2008 [107]. The reaction proceeded via C-H alkenylation/electrocyclization/aromatization sequence through dihydropyridine intermediates. The C-H activated complex was isolated and determination by X-ray analysis. Good yields of highly substituted pyridines were produced in one-pot manner (Scheme 3.50). 

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