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Direct Detection of the Acetate-forming Activity of the Enzyme Acetate Kinase
Published on: December 19, 2011
A spectrophotometric method to assay epoxide hydrolase activity
T Bhatnagar1, K M Manoj, J C Baratti
1Biocatalyse et Chimie Fine, Université de la Méditerranée, Faculté des Sciences de Luminy, case 901, 13288 cedex 9, Marseille, France.
Journal of Biochemical and Biophysical Methods
|November 21, 2001
Summary
A new spectrophotometric assay effectively measures Aspergillus niger epoxide hydrolase (EH) activity using p-nitrostyrene oxide. This method provides a fast, reproducible, and accurate evaluation of EH kinetics.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Enzyme assays are crucial for understanding enzyme kinetics and function.
- Accurate and efficient assays are needed for epoxide hydrolase (EH) characterization.
Purpose of the Study:
- To develop and validate a novel spectrophotometric assay for Aspergillus niger epoxide hydrolase (EH) activity.
- To enable routine, fast, and accurate evaluation of EH activity and kinetic parameters.
Main Methods:
- Spectrophotometric assay using (rac) p-nitrostyrene oxide (pNSO) as substrate.
- Extraction of unreacted pNSO with chloroform, quantifying product p-nitrostyrene diol (pNSD) by UV absorbance at 280 nm.
- Kinetic analysis and comparison with High-Performance Liquid Chromatography (HPLC) assay.
Main Results:
- Chloroform effectively extracted pNSO (>99%) with minimal pNSD co-extraction (32%).
- The assay demonstrated linearity with enzyme concentration and reaction time (first 10 min).
- The Michaelis constant (K(M)) for (rac) pNSO hydrolysis was determined as 1.1 mM, consistent with previous findings.
Conclusions:
- The developed spectrophotometric assay is a reliable, rapid, and reproducible method for assessing EH activity.
- This assay facilitates routine kinetic parameter determination for A. niger epoxide hydrolase.
- The method offers a valuable alternative to existing assays like HPLC for EH activity evaluation.
