Related Experiment Video
Updated: Jul 12, 2026
Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases
Published on: February 24, 2018
NADH-Dependent Oxidoreductase Activity Assay of OsAIM1 Using a Microplate Reader
Yuan Hu1, Song Cui1, Haishen Li1
1State Key Laboratory for Crop Genetics & Germplasm Enhancement and Utilization, Zhongshan Biological Breeding Laboratory, National Observation and Research Station of Rice Germplasm Resources, Nanjing Agricultural University, Nanjing, China.
A new spectrophotometric assay quantifies enzyme activity by monitoring NADH absorbance changes. This high-throughput method enables real-time kinetic analysis of NADH-dependent enzymes, crucial for biochemical characterization.
Area of Science:
- Biochemistry
- Enzymology
- Plant Science
Background:
- Peroxisomal β-oxidation is vital for jasmonic acid biosynthesis.
- Accurate enzyme characterization is needed for genetic variant comparisons.
- Current assays lack high-throughput and real-time kinetic measurement capabilities.
Purpose of the Study:
- To develop a novel spectrophotometric assay for quantifying enzymatic activity.
- To enable real-time kinetic measurements of NADH-dependent enzymes.
- To provide a standardized, high-throughput method for enzyme characterization.
Main Methods:
- Developed a spectrophotometric assay using a plate reader to measure NADH absorbance decrease at 340 nm.
- Utilized a 96-well plate format for reaction systems.
- Optimized reaction components, protein concentrations, and data processing for accuracy and reproducibility.
Main Results:
- The assay accurately determines OsAIM1 enzymatic activity in real-time.
- Achieved linearity, reproducibility, and quantitative accuracy through systematic optimization.
- Demonstrated suitability for calculating reaction rates and specific activity.
Conclusions:
- The developed assay is simple, requires low reaction volumes, and offers high throughput.
- This method is ideal for functional and kinetic analysis of NADH-dependent enzymes.
- Facilitates efficient comparison of enzyme activity across different samples or genetic variants.
