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An ultrafiltration assay for lysyl oxidase
1Department of Biochemistry, University of Edinburgh Medical School, United Kingdom.
Analytical Biochemistry
|March 1, 1990
Summary
This study introduces a modified lysyl oxidase assay using ultrafiltration, simplifying radioactive product separation. The new method offers convenience and avoids radioactive glassware decontamination for accurate enzyme activity measurement.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Lysyl oxidase is crucial for connective tissue formation.
- Accurate measurement of lysyl oxidase activity is essential for research.
Purpose of the Study:
- To describe a modified microdistillation assay for lysyl oxidase.
- To enhance assay sensitivity and substrate efficiency.
- To simplify the assay procedure and radioactive waste management.
Main Methods:
- Utilized Amicon C-10 microconcentrators for ultrafiltration to separate 3H-labeled products.
- Employed a [4,5-3H]-lysine-labeled elastin substrate.
- Determined enzyme activity via scintillation counting of the ultrafiltrate, with subtraction of inhibitor control radioactivity.
Main Results:
- Optimized conditions for assay sensitivity and substrate utilization.
- Demonstrated linear inhibition of lysyl oxidase activity up to 1 M urea.
- Showed comparable results between ultrafiltration and microdistillation methods.
- Validated direct assaying of samples in 6 M urea without dialysis.
Conclusions:
- The modified assay is simple, convenient, and efficient.
- Eliminates the need for radioactive glassware decontamination.
- Provides a reliable method for measuring lysyl oxidase activity.
