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Combinatorial screening of enzyme activity by using multiplexed capillary electrophoresis
1Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, 50011, USA.
Analytical Chemistry
|August 12, 2000
Summary
This study presents a novel method for high-throughput enzyme screening using 96-capillary array electrophoresis. Lactate dehydrogenase (LDH) enzyme activity was optimized at pH 7 for efficient drug discovery and diagnostics.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Enzyme Kinetics
Background:
- Enzyme activity screening is crucial for drug discovery and diagnostics.
- Current methods can be time-consuming and lack comprehensive throughput.
- Developing efficient, high-throughput screening platforms is essential.
Purpose of the Study:
- To develop and validate a novel method for efficient and comprehensive enzyme activity screening.
- To demonstrate the utility of the developed platform for enzyme characterization.
Main Methods:
- Utilized a combinatorial array of 96 reaction microvials for parallel screening.
- Employed 96-capillary array electrophoresis coupled with multiplexed absorption detection for quantitation.
- Analyzed reaction kinetics at well-defined time intervals.
Main Results:
- Successfully screened enzyme activity in a 96-well format.
- Capillary electrophoresis provided high resolution for product-reactant separation.
- Absorption detection demonstrated universal applicability for screening.
- Lactate dehydrogenase (LDH) catalytic activity was confirmed highest at pH 7 for NADH to NAD+ conversion.
Conclusions:
- The developed scheme enables efficient and comprehensive enzyme activity screening.
- This platform is suitable for high-throughput drug discovery, clinical diagnosis, substrate binding studies, and combinatorial synthesis.