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Single-Molecule Oxidoreductase Activity Analysis for Activity-Based Diagnosis Based on Proteoform Alterations
Mayano Minoda1, Junpei Hatakeyama2, Norimichi Nagano1
1Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Journal of the American Chemical Society
|January 6, 2025
Summary
We created a new assay to measure enzyme activity in single molecules, useful for diagnosing conditions like liver damage in patients. This method detects specific enzyme changes in blood and cerebrospinal fluid.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Biomedical diagnostics
Background:
- NAD(P)+-dependent oxidoreductases play crucial roles in cellular metabolism.
- Accurate measurement of enzyme activity is vital for disease diagnosis.
- Existing methods may lack sensitivity or require large sample volumes.
Purpose of the Study:
- To develop a novel single-molecule enzyme activity assay platform.
- To detect activities of NAD(P)+-dependent oxidoreductases.
- To explore the diagnostic potential in clinical samples.
Main Methods:
- Development of a single-molecule enzyme activity assay.
- Utilized a new NAD(P)H-responsive fluorogenic probe.
- Optimized for microdevice-based fluorometric detection.
- Applied to analyze enzyme activities in blood and cerebrospinal fluid (CSF).
Main Results:
- Successfully detected activities of lactate dehydrogenase, glucose-6-phosphate dehydrogenase, and hexokinases.
- Demonstrated feasibility in complex biological matrices like blood and CSF.
- Identified altered enzyme activity profiles in liver damage associated with brain tumors.
Conclusions:
- The developed platform enables sensitive single-molecule detection of enzyme activities.
- It shows promise for activity-based diagnosis using blood and CSF.
- Potential application in diagnosing conditions like liver damage in specific patient populations.
