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Lectin-glycoenzyme column chromatography monitored by enzyme flow microcalorimetry
D Mislovicová1, M Chudinová, A Vikartovská
1Institute of Chemistry, Slovak Academy of Sciences, Slovak Republic.
Journal of Chromatography. A
|January 26, 1996
Summary
Flow microcalorimetry effectively monitors immobilized enzyme activity during lectin affinity chromatography. This method allows direct assessment of enzyme binding and stability, offering a novel approach for glycoenzyme analysis.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Chromatography
Background:
- Lectin affinity chromatography is crucial for purifying glycoenzymes.
- Monitoring enzyme activity and binding during chromatography is essential for process optimization.
- Enzyme thermistors offer sensitive detection of enzyme activity.
Purpose of the Study:
- To develop and validate a flow microcalorimetric method for monitoring lectin affinity chromatography of immobilized enzymes.
- To investigate the direct determination of bound enzyme catalytic activity using microcalorimetry.
- To assess the operational parameters and storage stability of the affinity sorbent.
Main Methods:
- Flow microcalorimetry utilizing an enzyme thermistor.
- Lectin affinity chromatography of immobilized invertase on Concanavalin A-bead cellulose.
- Post-column analysis using the enzyme thermistor method.
Main Results:
- Flow microcalorimetry directly correlated immobilized invertase catalytic activity with the thermometric value delta Tmax.
- The method successfully monitored enzyme adsorption and desorption in the chromatography column.
- The affinity matrix showed satisfactory storage stability but decreased binding ability with repeated use.
Conclusions:
- Flow microcalorimetry provides a valid and direct method for monitoring lectin affinity chromatography of glycoenzymes.
- The technique allows for real-time assessment of enzyme binding and activity.
- Understanding sorbent stability is key for optimizing chromatographic processes.