Use of a 96-well microplate reader for measuring routine enzyme activities.
A method is described for the routine determination of the rate of colorimetric enzyme reactions using a 96-well microtiter plate reader commonly used in immunoassay. This approach is illustrated by monitoring esterase activity using three common products: release of thiol, release of ethanol, and release of p-nitrophenylate ion. Examples include monitorings of the rate of hydrolysis of acetylthiocholine iodide by eel acetylcholinesterase and the rate of hydrolysis of malathion and nonconventional esters such as O-methyl, O-ethyl, and O-isobutyl carbonates of p-nitrophenol by commercial porcine liver carboxylesterase. Data obtained from the plate reader were compared to those obtained, under similar conditions, in a conventional spectrophotometer. Absorbance measurements made in both machines on the same solution, as well as absorbance changes measured over time, were similar. The use of the 96-well plate format tremendously increased the number of enzyme assays carried out per person and the interface with a personal computer allowed rapid manipulation of the absorbance values to calculate the desired rate data. This approach should be generally applicable to many routine colorimetric assays in the research laboratory.
Department of Entomology, University of California, Davis 95616.
M B
MB
Ashour
S J
SJ
Gee
B D
BD
Hammock
D000110:Acetylcholinesterase / Q000032:analysis; D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000032:analysis; D004524:Eels; D004798:Enzymes / Q000032:analysis*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009596:Nitrophenols / Q000378:metabolism; D013552:Swine
D004798:Enzymes; D009596:Nitrophenols; D002265:Carboxylic Ester Hydrolases; D000110:Acetylcholinesterase; C024836:4-nitrophenol