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Peroxidase active site activity assay.

Kelsey C Duggan¹, Joel Musee, Lawrence J Marnett

¹A.B. Hancock Jr. Memorial Laboratory for Cancer Research, Department of Biochemistry, Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, TN, USA.

Methods in Molecular Biology (Clifton, N.J.)

|July 21, 2010

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This study details assays for cyclooxygenase enzyme peroxidase activity. New spectrophotometric and HPLC methods allow for accurate measurement of key enzymatic reactions involved in prostaglandin synthesis.

Area of Science:

Biochemistry
Enzymology

Background:

Cyclooxygenase (COX) enzymes possess distinct cyclooxygenase and peroxidase active sites.
Peroxidase activity involves heme reduction and tyrosyl radical formation, crucial for activating the cyclooxygenase site.
This process initiates the conversion of arachidonic acid to prostaglandin precursors like PGG(2).

Purpose of the Study:

To describe novel assay methods for quantifying cyclooxygenase enzyme peroxidase activity.
To provide tools for researchers studying prostaglandin and thromboxane biosynthesis pathways.

Main Methods:

Development of a spectrophotometric assay measuring the oxidation of 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as an indirect measure of peroxidase activity.
Establishment of a high-performance liquid chromatography (HPLC) method to directly measure the reduction of 5-phenyl-4-pentyl hydroperoxide (PPHP) to its alcohol.

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Main Results:

The spectrophotometric assay offers continuous monitoring of peroxidase activity.
The HPLC method provides a discontinuous but direct measurement of hydroperoxide reduction.
Both methods enable accurate quantification of cyclooxygenase peroxidase function.

Conclusions:

The described assays provide valuable tools for investigating cyclooxygenase enzyme kinetics and function.
Accurate measurement of peroxidase activity is essential for understanding the biosynthesis of prostaglandins and thromboxanes.
These methods facilitate research into the roles of cyclooxygenase enzymes in physiological and pathological processes.