Use of a 96-well microplate reader for measuring routine enzyme activities

Mohamed Bassem A Ashour, Shirley J. Gee, Bruce D. Hammock

Research output: Contribution to journalArticle

67 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)353-360
Number of pages8
JournalAnalytical Biochemistry
Volume166
Issue number2
DOIs
StatePublished - Nov 1 1987

Keywords

  • 96-well plate reader
  • carboxylesterases
  • cholinesterase
  • enzyme assay
  • enzyme rates
  • microtiter plates
  • nitrophenyl carbonates
  • semiautomated

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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