Peroxidative oxidation of leuco-dichlorofluorescein by prostaglandin H synthase in prostaglandin biosynthesis from polyunsaturated fatty acids

doi:10.1016/0005-2760(95)00188-3

Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism

Volume 1299, Issue 1, 5 January 1996, Pages 47-53

Biochimica et Biophy...

https://doi.org/10.1016/0005-2760(95)00188-3 Get rights and content

Abstract

Prostaglandin H synthase can oxidize arachidonic acid with leuco-dichlorofluorescein as reducing cosubstrate. Addition of 0.5 mM phenol increases the oxidation of leuco-dichlorofluorescein 5-fold, probably by acting as a cyclic intermediate in the oxidation. Tetramethyl-p-phenylenediamine is also oxidized as cosubstrate. Its oxidation is not influenced by phenol. A stoichiometry of close to one mole of tetramethyl-p-phenylenediamine or leuco-dichlorofluorescein consumed per mole of arachidonic acid was found in the initial phase of the reaction. In the presence of phenol + leuco-dichlorofluorescein, the oxidation rate of arachidonic acid is about 40% lower than with phenol alone as cosubstrate. Since dichlorofluorescein has a molar extinction coefficient of 91 · 10³ at 502 nm, the oxidation of less than 1 μM leuco-dichlorofluorescein can be detected spectrophotometrically. The rate of extinction change with leuco-dichlorofluorescein (at 502 nm) is about 4-fold more rapid than with tetramethyl-p-phenylenediamine (at 611 nm). With this spectrophotometric assay we have confirmed that arachidonic acid, linolenic acid, adrenic acid, γ-linolenic acid, eicosapentaenoic acid, are substrates for prostaglandin H synthase with decreasing reaction rates in the mentioned order. The same order of reaction rates were found when oxygen consumption was measured. The assay also shows that docosahexaenoic acid is substrate for the enzyme. The reaction rate of the enzyme evidently is decreased both by a n − 3 double bond and by deviation from a 20 carbon chain length of the fatty acid substrate.

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Regular paperPeroxidative oxidation of leuco-dichlorofluorescein by prostaglandin H synthase in prostaglandin biosynthesis from polyunsaturated fatty acids

Abstract

Biochim. Biophys. Acta

J. Biol. Chem.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Progr. Lipid Res.

J. Biol. Chem.

Biochim. Biophys. Acta

Prostaglandins

Prostaglandins

Anal. Biochem.

Prostaglandins

Prostaglandins

Anal. Biochem.

J. Biol. Chem.

Anal. Biochem.

Prostaglandins

Regular paper
Peroxidative oxidation of leuco-dichlorofluorescein by prostaglandin H synthase in prostaglandin biosynthesis from polyunsaturated fatty acids