Ellman method

Collect fractions of about 1 mL and determine their enzymatic activity using the Ellman method (see Note 3) Pool the fractions corresponding to the G4 form (second major peak) and determine the AChE concentration of the pool enzymatically. G4-SMCC can be used immediately It can also be kept frozen at-80°C for at least 6 mo. 

Henderson, J., M. O Malley, J. Sanborn, M. Verder-Carlos and B. Wilson (1998). Comparison of Acetylcholinesterase Assays Run Under Conditions Specified by the Standard Ellman Method and Conditions Specified by a Commercial Cholinesterase Reagent Kit, Department of Pesticide Regulation, Worker Health and Safety Branch Report HS-1752, Sacramento, CA, USA. 

Transient activity during the first 5-10 min of DTNB by using the Ellman method. Source Loof (1992). 

The Ellman method for sulfinyl ketimine synthesis would similarly suffer, but perhaps can be alleviated by using the Lewis acids B(OiPr)3 or Al(OiPr)3. 

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. 

OPs and CMs are used as pesticides, and some OPs are also potential warfare nerve agents. Pesticides are used worldwide, and warfare agcni.s may also be used extensively. To detect absorption of anticholinesterases, a standardized protocol for measuring AChE and BuChE activities in human blood is needed that is simple in technical terms but reliable and well reproducible. This would enable a better comparison of data from different laboratories and an exchange of samples for analysis and confirmation of results. It seems that a protocol based on the Ellman method would meet the outlined requirements, but further studies including interlaboratory quality control are required, particularly when field methods are developed. 

Reiner, E Skrinjarie-Spoljar, M.. Sinko, G., and Simeon-Rudolf, V, (2000). Comparison of protocols for measuring of human blood cholinesterases by the Ellman method. Arh. His. Rada. Toksikol. 51,13-18,... 

The activities of two enzymes have been used as biomarkers of effects for OPs, namely acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase, sometimes known as pseudocholinesterase (EC 3.1.1.8). The structure and function of these enzymes has been reviewed. " In humans the former is present in red blood cells and the latter in plasma, but such distribution is not true of all species. In dogs, both enzymes are present in plasma with a ratio of butyrylcholinesterase to acetylcholinesterase of 7 1, while in the rat, plasma cholinesterase activity comprises more acetylcholinesterase with a butyrylcholinesterase to acetylcholinesterase activity of 1 3 in males and 2 1 in females in neither blood compartment are the functions of the enzymes fully understood.Because of the possibility of confusion, the terms plasma cholinesterase and erythrocyte cholinesterase as synonyms for butyrylcholinesterase and acetylcholinesterase are to be deprecated, especially when used of enzymes in animals where serious confusion may result. It is often necessary to look in detail at animal studies to see what activity has been measured in each matrix. In particular, it is necessary to look at the substrate(s) used in the assay together with any inhibitors used. Methods for measuring acetylcholinesterase have been reviewed and acetylcholinesterase and butyrylcholinesterase activities can be measured separately. In almost all cases it is the enzyme activity, rather than protein concentration, that is measured and many of the procedures used are variants of the Ellman method. Correct storage of blood samples is important as reactivation of inhibited enzymes ex vivo can occur. 

---