A novel electron paramagnetic resonance-based assay for prostaglandin H synthase-1 activity
<p>Abstract</p> <p>Background</p> <p>Prostaglandin H<sub>2 </sub>synthase (PGHS) is the enzyme that catalyses the two-stage conversion of arachidonic acid to prostaglandin H<sub>2 </sub>(PGH<sub>2</sub>) prior to formation of prostano...
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| Main Authors: | , , , |
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| Format: | Book |
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BMC,
2006-09-01T00:00:00Z.
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| Online Access: | Connect to this object online. |
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| Summary: | <p>Abstract</p> <p>Background</p> <p>Prostaglandin H<sub>2 </sub>synthase (PGHS) is the enzyme that catalyses the two-stage conversion of arachidonic acid to prostaglandin H<sub>2 </sub>(PGH<sub>2</sub>) prior to formation of prostanoids that are important in inflammation. PGHS isozymes (-1 and -2) are the target for nonsteroidal anti-inflammatory drugs (NSAIDs).</p> <p>Given the rekindled interest in specific anti-inflammatory PGHS inhibitors with reduced unwanted side effects, it is of paramount importance that there are reliable and efficient techniques to test new inhibitors. Here, we describe a novel <it>in vitro </it>electron paramagnetic resonance (EPR)-based assay for measuring the activity of PGHS-1.</p> <p>Methods</p> <p>We validated a novel <it>in vitro </it>PGHS-1 activity assay based on the oxidation of spin-trap agent, 1-hydroxy-3-carboxy-pyrrolidine (CPH) to 3-carboxy-proxy (CP) under the action of the peroxidase element of PGHS-1. This quantifiable spin-adduct, CP, yields a characteristic 3-line electron paramagnetic (EPR) spectrum.</p> <p>Results</p> <p>The assay is simple, reproducible and facilitates rapid screening of inhibitors of PGHS-1. Aspirin (100 μM, 1 mM) caused significant inhibition of spin-adduct formation (72 ± 11 and 100 ± 16% inhibition of control respectively; P < 0.05). Indomethacin (100 μM) also abolished the signal (114 ± 10% inhibition of control; P < 0.01). SA and the PGHS-2-selective inhibitor, NS398, failed to significantly inhibit spin-adduct generation (P > 0.05).</p> <p>Conclusion</p> <p>We have demonstrated and validated a simple, reproducible, quick and specific assay for detecting PGHS-1 activity and inhibition. The EPR-based assay described represents a novel approach to measuring PGHS activity and provides a viable and competitive alternative to existing assays.</p> |
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| Item Description: | 10.1186/1476-9255-3-12 1476-9255 |