A Radiochemical Assay for COX Enzymes
Full Papers
Development of a Radiochemical Cyclooxygenase-1 and -2 in Vitro Assay for
Identification of Natural Products as Inhibitors of Prostaglandin Biosynthesis
Ylva Noreen,† Therese Ringbom,† Premila Perera,† Helena Danielson,‡ and Lars Bohlin*,†
Division of Pharmacognosy, Department of Pharmacy, Biomedical Centre, Uppsala University, Box 579,
S-751 23 Uppsala, Sweden, and Department of Biochemistry, Biomedical Centre, Uppsala University, Box 576,
S-751 23 Uppsala, Sweden
Received July 18, 1997X
A radiochemical enzyme assay for studying cyclooxygenase (COX)-catalyzed prostaglandin biosynthesis in vitro was optimized with respect to both COX-1 and COX-2 activity. The assay can be used to assess the relative selectivity of plant-derived inhibitors on COX-1 and COX-2.
Assay conditions were optimized for both enzymes with respect to concentration of cofactors
(l-epinephrine, reduced glutathione, and hematin), activation time (enzyme and cofactors), reaction time, and pH. Moreover, the kinetic parameters, Km and Kcat., of both enzymes were estimated. Five COX inhibitors were used to validate the assay, indomethacin, aspirin, naproxen, ibuprofen, and the arylsulfonamide NS-398, all with different COX selectivity and time dependency. Time-dependent inhibition was determined by comparing the inhibition, with and without preincubation of enzyme and inhibitor. Two flavonoids, (+)-catechin and quercitrin, were examined with respect to inhibition of COX-catalyzed prostaglandin biosynthesis.
(+)-Catechin showed equal inhibitory effects on the two enzymes. Quercitrin was found to be inactive toward both COX-1- and COX-2-catalyzed prostaglandin biosynthesis. The optimization procedure resulted in a considerable reduction of the amount of enzyme required for adequate prostglandin biosynthesis and a reliable method suited to evaluate natural products on inhibition of COX-2-catalyzed prostaglandin biosynthesis, as well as on COX-1.
The enzyme
Development of a Radiochemical Cyclooxygenase-1 and -2 in Vitro Assay for
Identification of Natural Products as Inhibitors of Prostaglandin Biosynthesis
Ylva Noreen,† Therese Ringbom,† Premila Perera,† Helena Danielson,‡ and Lars Bohlin*,†
Division of Pharmacognosy, Department of Pharmacy, Biomedical Centre, Uppsala University, Box 579,
S-751 23 Uppsala, Sweden, and Department of Biochemistry, Biomedical Centre, Uppsala University, Box 576,
S-751 23 Uppsala, Sweden
Received July 18, 1997X
A radiochemical enzyme assay for studying cyclooxygenase (COX)-catalyzed prostaglandin biosynthesis in vitro was optimized with respect to both COX-1 and COX-2 activity. The assay can be used to assess the relative selectivity of plant-derived inhibitors on COX-1 and COX-2.
Assay conditions were optimized for both enzymes with respect to concentration of cofactors
(l-epinephrine, reduced glutathione, and hematin), activation time (enzyme and cofactors), reaction time, and pH. Moreover, the kinetic parameters, Km and Kcat., of both enzymes were estimated. Five COX inhibitors were used to validate the assay, indomethacin, aspirin, naproxen, ibuprofen, and the arylsulfonamide NS-398, all with different COX selectivity and time dependency. Time-dependent inhibition was determined by comparing the inhibition, with and without preincubation of enzyme and inhibitor. Two flavonoids, (+)-catechin and quercitrin, were examined with respect to inhibition of COX-catalyzed prostaglandin biosynthesis.
(+)-Catechin showed equal inhibitory effects on the two enzymes. Quercitrin was found to be inactive toward both COX-1- and COX-2-catalyzed prostaglandin biosynthesis. The optimization procedure resulted in a considerable reduction of the amount of enzyme required for adequate prostglandin biosynthesis and a reliable method suited to evaluate natural products on inhibition of COX-2-catalyzed prostaglandin biosynthesis, as well as on COX-1.
The enzyme
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