Determination of Arachidonic Acid Based on the Prostaglandin H Synthase Catalyzed Reaction

• Published in: Applied biochemistry and biotechnology 2000-07, Vol.88 (1-3), p.33-44
• Main Authors: Ilyina, Anna D, Martínez Hernández, José L, Badillo, César Estrada, Sena Maldonado, María G, Galindo, Sara Carrillo, González, María Hernández, Martínez, Jesús Rodríguez
• Format: Article
• Language: English
• Subjects: Acids; arachidonic acid; Biochemistry; Buffers; Catalysis; Chemiluminescence; Hydrogen peroxide; prostaglandin H synthase; Studies
• ISSN: 0273-2289; 0273-2289; 1559-0291
• DOI: 10.1385/ABAB:88:1-3:033

This article presents a novel method of arachidonic acid (AA) determination based on the reaction catalyzed by prostaglandin H synthase (PGHs). The deoxygenated and nondeoxygenated (as control) buffers were used to obtain the PGHs preparations from bovine vesicular glands by two different methods. The higher specific activity was observed for solubilized preparations obtained by ultracentrifugation and deoxygenated buffers. The preparations obtained by Ca^sup 2+^ treatment demonstrated higher stability of PGHs during its storage at -15°C. To record the initial rate of AA transformation, a spectrophotometric assay of PGHs cyclo-oxygenase and peroxidase activities was developed using adrenaline and ABTS as electron donors. No oxidation of ABTS was observed in the reaction of AA transformation catalyzed by the PGHs from bovinevesicular glands. However, this electron donor was successfully used in the reaction catalyzed by PGHs from sheep vesicular glands. No chemiluminescence was recorded in the reaction of AA transformation catalyzed by PGHs from bovine vesicular glands in the presence of luminol. The chemiluminescent intensity was measured after addition of hydrogen peroxide allowing quantitative assay of AA to be performed.[PUBLICATION ABSTRACT]