A novel electron paramagnetic resonance-based assay for prostaglandin H synthase-1 activity

• Main Authors: Rossi Adriano G, McClure Danny, Turnbull Catriona M, Megson Ian L
• Format: Article
• Language: English
• Published: BMC 2006-09-01
• Series: Journal of Inflammation
• Online Access: http://www.journal-inflammation.com/content/3/1/12

<p>Abstract</p> <p>Background</p> <p>Prostaglandin H₂synthase (PGHS) is the enzyme that catalyses the two-stage conversion of arachidonic acid to prostaglandin H₂(PGH₂) 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>