PEROXIDATIVE OXIDATION OF LEUCO-DICHLOROFLUORESCEIN BY PROSTAGLANDIN-H SYNTHASE IN PROSTAGLANDIN BIOSYNTHESIS FROM POLYUNSATURATED FATTY-ACIDS

Prostaglandin H synthase can oxidize arachidonic acid with leuco-dichl orofluorescein as reducing cosubstrate. Addition of 0.5 mM phenol incr eases the oxidation of leuco-dichlorofluorescein to dichlorofluorescei n 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 c onsumed 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 ph enol alone as cosubstrate. Since dichlorofluorescein has a molar extin ction coefficient of 91 . 10(3) at 502 nm, the oxidation of less than 1 mu M leuco-dichlorofluorescein can be detected spectrophotometricall y. The rate of extinction change with leuco-dichlorofluorescein (at 50 2 nm) is about 4-fold more rapid than with tetramethyl-p-phenylenediam ine (at 611 nm). With this spectrophotometric assay we have confirmed that arachidonic acid, linolenic acid, adrenic acid, gamma-linolenic a cid, eicosapentaenoic acid, are substrates for prostalgandin H synthas e with decreasing reaction rates in the mentioned order. The same orde r of reaction rates were found when oxygen consumption was measured. T he assay also shows that docosahexaenoic acid is substrate for the enz yme. 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 th e fatty acid substrate.