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A continuous fluorometric assay for phospholipases using polymerized mixed liposomes

S K Wu1, W Cho

  • 1Department of Chemistry, University of Illinois at Chicago 60607-7061.

Analytical Biochemistry
|August 15, 1994
PubMed
Summary

A new fluorometric assay uses stable polymerized liposomes to detect phospholipase A2, C, and D activity. This method offers sensitive and rapid enzyme detection with broad applicability.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Analytical Chemistry

Background:

  • Phospholipases A2, C, and D are crucial enzymes involved in various biological processes.
  • Existing assays for these enzymes often lack sensitivity, stability, or versatility.
  • Development of a robust and adaptable assay is needed for accurate phospholipase activity measurement.

Purpose of the Study:

  • To develop a versatile and continuous fluorometric assay for phospholipases A2, C, and D.
  • To utilize novel polymerized mixed liposomes incorporating pyrene-containing phospholipids for enhanced detection.
  • To optimize assay conditions and validate its performance across different phospholipase sources.

Main Methods:

  • Synthesized polymerized mixed liposomes incorporating pyrene-labeled phospholipids.

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  • Employed specific pyrene-containing phospholipids for phospholipase A2 and C/D assays.
  • Monitored enzyme activity via fluorescence increase upon hydrolysis and pyrene moiety displacement.
  • Optimized assay parameters by adjusting liposome surface charge and phospholipid structure.
  • Determined enzyme activity and specific activity using a fluorescence plate reader.

    • Main Results:

    • The assay demonstrated selective and rapid hydrolysis of pyrene-containing phospholipids by all tested phospholipases.
    • A significant increase in fluorescence emission at 380 nm correlated with enzyme activity.
    • Phospholipase activity showed a linear response to enzyme concentration (0.1-50 ng).
    • Specific activity measurements for various phospholipases ranged from 0.5 to 100 mumol/min/mg.
    • Polymerized liposomes exhibited exceptional stability against chemical and physical degradation.

    Conclusions:

    • A versatile, continuous, and sensitive fluorometric assay for phospholipases A2, C, and D has been successfully developed.
    • The assay utilizes stable polymerized mixed liposomes, enabling rapid and selective enzyme detection.
    • This method allows for direct measurement of enzyme-liposome complex dissociation constants due to the inert polymerized matrix.