A rapid, simple radiometric assay for cholinesterase, suitable for multiple determinations

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Abstract

A rapid and simple radiometric assay for cholinesterase, suitable for multiple determinations, has been developed. (3H-acetyl) choline is enzymatically hydrolyzed in a small reaction volume in a scintillation vial. The released [3H]acetate is then extracted into a toluene-based scintillator added directly to the vial, without removing the reaction volume. The extracted [3H]acetate counts efficiently, but the unhydrolyzed [3H]acetylcholine remains unextracted in the small aqueous reaction volume, from which its weak β-particles of decay do not escape to excite the scintillator. The assay is highly reproducible, quite sensitive, and useful for applications in which multiple samples must be quickly assayed.

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    The tubes containing the remainder of the diluted blood were then fixed onto a Nutating mixer and gently agitated overnight at room temperature prior to the 24 h sample harvest. At the time of ChE determination (Johnson and Russell, 1974), the treated blood samples were thawed at 4 °C and 30 μl aliquots were placed into 5 ml scintillation vials in test tube racks held on crushed ice. The samples were pre-incubated for 5 min in a 37 °C water bath prior to the initiation of the assay by the addition of radioactive substrate warmed to 37 °C.

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This work was supported by a Grant from the U. S. Public Health Service (NS 09654) and by a Sloan Foundation Neurosciences Research Grant to R.L.R.

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