Assay of COX-1 and COX-2 activity

Cyclooxygenase or Cox or Prostaglandin H synthase (PGHS) oxidises arachidonic acid to prostaglandin G2 using two molecules of dioxygen. Prostaglandin G2 is further metabolised by the same enzyme to prostaglandin H2 in the presence of a reducing substrate such as phenol or epinephrine. Three methods can be used for measuring the activity of PGHS. The first assay relies on the measurement of the rate of oxygen consumption using a Clark oxygen electrode. The second assay measures the peroxidase activity of PGHS using a Spectrophotometer. The third assay relies on the measurement Prostaglandins (PGF2a and PGE2) derived from PGH2 using EIA. Chemical conversion of PGH2 to PGF2a is facilitated by the addition of stannous chloride, whereas PGE isomerase is required for the conversion of PGH2 to PGE2. The three methods are described in detail below.

Definition of enzyme units : One unit of prostaglandin H synthase is defined as the amount of enzyme that consumes one nanomole of dissolved oxygen per minute at 37ºC under the conditions of the assay described.

Preparation of stock substrate (arachidonic acid) solution : A stock solution of sodium or potassium arachidonate (6.5 mM) for utilization in either assay is prepared as follows: To 2 mg of arachidonic acid in 100 µl of ethanol, 100 µl of 0.1 M sodium (or potassium) hydroxide is added and mixed well. The mixture is cooled on ice and 800 µl of metal ion free water is added and mixed well to obtain 6.5 mM arachidonate solution. This solution, if protected from light and stored at 0-5ºC, is stable for at least 12 hours.

Oxygraph assay

Measurement of the rate of oxygen uptake is a standard assay procedure used for any enzyme that uses molecular oxygen as a cosubstrate. This assay requires about 50-100 units of enzyme per assay.

Peroxidase assay

Peroxidase activity of PGHS is determined by following the oxidation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) at 37°C using hydrogen peroxide or arachidonate as the substrate. The reaction mixture (3 ml) contained 0.1 M Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM hematin, 0.2 mM TMPD, 100 mM substrate (hydrogen peroxide or arachidonate), and PGHS. The reaction is initiated with substrate. Increase in absorbance at 611 nm is recorded on a Beckman DU-70 Spectrophotometer. With hydrogen peroxide as substrate the reaction is linear for at least 2 min, whereas with arachidonate as substrate, the linearity ceases after 10 sec. Peroxidase activity is calculated based on an extinction coefficient of 13,500 M-1cm-1 and a stoichiometry of 2 moles of TMPD oxidized per mole of hydroperoxide reduced.

Product assay

Chemical conversion of PGH2 to Prostaglandin F2alpha (PGF2alpha) using stannous chloride provides an alternative method of measuring PGHS activity. PGF2acan be measured by EIA with very high sensitivity (pg/ml). As a result, this method requires much less enzyme for each assay (5-10 units). The conversion of PGH2 to PGF2a is usually quantitative but other decomposition products such as PGE2 may also be produced during the reaction and thus we recommend using the Prostaglandin Screen EIA Assay (Cat # 514012). The product assay is a suggested method of measuring PGHS activity and must be standardized by each user.

The reaction described below is for an endpoint assay (i.e. single point measurement) of the enzyme. The amount of product formed in the time prior to quenching the reaction will be a reasonable measure of the enzyme activity provided the amount of substrate utilized during the reaction is a small fraction (<10%) of the initial substrate concentration.

Details of the reaction:

Buffer: 0.1 M Tris-HCl (pH 8.0) containing 1 mM EDTA, 0.5 mM phenol, and 1 mM Hematin;
Substrate: 20 µM arachidonic acid (final concentration);
Temperature: 37°C (buffer pre-equilibrated to 37°C before the addition of enzyme);
Incubation time: 0.5-1.0 minutes after the addition of the enzyme;
SnCl2 solution: 100 mg/ml solution in 0.1 M HCl.

To 5-10 units of PGHS in a test tube enough buffer is added to bring the volume to 2 ml. The reaction is initiated by addition of 6 µl of stock arachidonic acid. The reaction is allowed to proceed for precisely 2 minutes upon which the reaction is quenched by addition of 50 µl of stannous chloride solution. The reaction is allowed to proceed for an additional 10 minutes. Dilute the solution further (serial dilutions ranging from approximately 1,000 to 100,000 fold) with EIA Buffer and measure the Prostaglandins by EIA.

(Note: Any PGHS inhibitors used in the assay may interfere with the enzyme immunoassay. It is imperative that the assay be standardised by the user to account for any such interferences. Also, make sure to do a negative control (assay without enzyme).

| Back to Scientific Section Page |