Effect of simultaneous incubation of COX-1 or COX-2 with ONOO−and arachidonic acid on PGH2 formation
| ONOO− | PGH2 | PGH2 | ||
|---|---|---|---|---|
| COX-1 | COX-2 | COX-1 | COX-2 | |
| μM | ng | % | ||
| 0 | 242 ± 6 | 58.8 ± 0.8 | 100 | 100 |
| 50 | 234 ± 38 | 59 ± 0.6 | 96.7 ± 15.6 | 100.4 ± 10 |
| 100 | 300 ± 89 | 64.3 ± 25 | 123.8 ± 37 | 108.5 ± 42 |
COX-1 (45.14 U, 0.92 μg of protein) and COX-2 (23.35 U, 3.36 μg of protein) per ml were incubated with ONOO− at pH 7.8 for 5 min then mixed with 10 μM 14C-labeled arachidonic acid, and incubated for 30 min. PGH2 was rapidly extracted with cold ethyl acetate and analyzed by a reversed-phase HPLC. The amount of PGH2 was calculated from the specific activity of arachidonic acid (54.6 mCi/mmol) and the area of PGH2 peak of radioactivity. Values are in ng/μg of protein and are averages of three measurements ± S.E.M.