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Assay strategies and methods for phospholipases.

L J Reynolds, W N Washburn, R A Deems

Methods in Enzymology

|January 1, 1991

Summary

Choosing the right enzyme assay depends on required sensitivity and whether it needs to be continuous. Radioactive TLC assays offer high accuracy and sensitivity, while pH-stat assays are suitable for continuous measurements when sensitivity is less critical.

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Area of Science:

Biochemistry
Enzymology

Background:

Selecting appropriate enzyme assays is crucial for accurate biochemical analysis.
Phospholipase assays have specific considerations including sensitivity, continuity, substrate, and detergent compatibility.

Purpose of the Study:

To review and compare various enzyme assay methodologies.
To identify the most suitable assays for phospholipase characterization.

Main Methods:

Discussion of general assay selection criteria (sensitivity, continuity, substrate availability, specificity).
Evaluation of commonly used phospholipase assays: radioactive TLC, pH-stat, radioactive E. coli, and spectrophotometric thiol assays.
Mention of emerging assays like SIBLINKS and fluorescent assays.

Main Results:

Radioactive TLC assay is highly accurate and sensitive but discontinuous, tedious, and expensive.
pH-stat assay is suitable for continuous measurements when high sensitivity is not required.
Radioactive E. coli assay offers a faster, less expensive alternative to TLC for specific applications.
Spectrophotometric thiol assay is convenient and reproducible but lacks commercial substrate.

Conclusions:

No single assay is universally ideal; the choice depends on specific experimental needs.
Ongoing research aims to develop a sensitive, continuous, and broadly applicable enzyme assay.

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