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A continuous fluorometric assay for leukotriene D4 hydrolase
I J White1, J Lawson, C H Williams
1School of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2 9JT, United Kingdom.
Analytical Biochemistry
|March 17, 1999
Summary
Researchers developed a novel fluorogenic substrate, epsilon-DNP-l-Lys-d-Amp, for assaying leukotriene D4 hydrolase (LTDase). This substrate is highly specific and effective for measuring LTDase activity in biological samples.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Leukotriene D4 hydrolase (LTDase) plays a role in inflammatory processes.
- Assaying LTDase activity is crucial for understanding its physiological and pathological functions.
- Existing methods for LTDase assay may lack specificity or sensitivity.
Purpose of the Study:
- To synthesize and evaluate a novel fluorogenic substrate for the specific assay of leukotriene D4 hydrolase (LTDase).
- To confirm the substrate's selectivity for LTDase in complex biological matrices.
Main Methods:
- Synthesis of the fluorogenic substrate epsilon-DNP-l-Lys-d-Amp, incorporating a quenched fluorophore and a D-amino acid.
- Enzyme kinetics studies using purified porcine kidney LTDase.
- Assay validation using porcine kidney microvillar membranes and the specific LTDase inhibitor cilastatin.
Main Results:
- The synthesized substrate, epsilon-DNP-l-Lys-d-Amp, demonstrated excellent substrate properties for LTDase.
- A Km value of 370 microM was determined for the substrate with purified LTDase.
- The substrate showed no self-quenching of product fluorescence and was selectively hydrolyzed by LTDase, as confirmed by inhibition with cilastatin.
Conclusions:
- Epsilon-DNP-l-Lys-d-Amp is a highly effective and selective fluorogenic substrate for the assay of leukotriene D4 hydrolase (LTDase).
- This new substrate enables accurate and sensitive measurement of LTDase activity, facilitating further research into its biological roles.