Singlet oxygen formation detected by low-level chemiluminescence during enzymatic reduction of prostaglandin G2 to H2.

Abstract

Addition of arachidonic acid to a suspension of ram vesicular gland microsomes or purified prostaglandin synthase, causes a rapid burst of light emission in the range 600-750 nm, as detected by single-photon counting. Maximal light emission intensity is obtained within 15-30 s after the addition of arachidonic acid and is followed by a rapid decay to the background level. The intensity of chemiluminescence is dependent on the amount of ram vesicular gland microsomes or isolated prostaglandin synthase and arachidonic acid concentration (Km about 6 microM). Spectral analysis of arachidonic acid-induced photoemission of isolated prostaglandin synthase in the range 600-750 nm showed two distinctive peaks at about 634 and 703 nm. The similar relative intensities of these peaks, along with the lower intensity at about 668 nm is indicative of singlet oxygen dimol emission. Chemiluminescence with arachidonate is enhanced by 1,4-diazabicyclo[2,2,2]octane and inhibited by azide, indomethacin, acetylsalicylic acid and beta-carotene. Cooxygenation substrates such as phenol, hydroquinone and reduced glutathione, inhibited the arachidonic acid-induced chemiluminescence. Dioxygen is a requirement for the observation of singlet oxygen dimol emission with arachidonic acid as a substrate for ram vesicular gland microsomes or purified prostaglandin synthase. However, when prostaglandin G2 is substituted for arachidonic acid, light emission is not dependent on oxygen. Thus, singlet oxygen can be formed in the dismutation reaction, 2 PGG2 leads to 2 PGH2 + 1O2, catalysed by prostaglandin hydroperoxidase.