Phospholipase C Assay

Method: Phospholipase activity values which are derived from titrimetric assay procedures can be quite dependent upon the source and the type of substrate, the preparation of the substrate emulsion, other components of the reaction mixture and the methodology and instrumentation used. Comparative investigation has resulted in the standardized method described below. Values obtained by this procedure have been found to be both reproducible and reliable. The reaction velocity is measured titrimetrically as the rate of hydrolysis of soybean lecithin emulsion. One unit is that amount of enzyme which releases one micromole of acid from soybean lecithin per minute at 25°C and pH 7.4 under the specified conditions.

Reagents

• 6 mM Imidazole*HCl buffer, pH 7.4, containing 2.2 mM calcium chloride
• 0.15 M Sodium chloride
• 0.01 N NaOH, standardized
• 2% Soybean lecithin. Prepare fresh daily by suspending 2.0 grams soybean lecithin in 100 ml of 0.15 M NaCl. Stir at moderate speed for 20 minutes on a magnetic stirrer. Sonicate for 10 minutes at maximum power.
• 0.64 M Calcium chloride

Enzyme

Dissolve at a concentration of 0.5 mg/ml in cold 6 mM imidazole buffer, pH 7.4 with 2.2 mM CaCl₂ just before the assay is performed.

Procedure

The titration can be measured with either an automatic titrator or with a laboratory pH meter. The reaction vessel should be maintained at 25°C.

Blank rate determination - Pipette into reaction vessel as follows:

2% Soybean lecithin	7.5 ml
0.64 M Calcium chloride	0.5 ml

Adjust pH to 7.4 and record the volume of the titrant required to maintain the pH at 7.4 for 4-5 minutes after a constant rate is obtained. Determine the "blank rate" as the volume (ml) of titrant added per minute from the final linear portion of the curve.

Sample determination - Add appropriately diluted enzyme to the above lecithin-CaCl₂ mixture. Record the amount of titrant required to maintain the pH at 7.4 for 4-5 minutes. Determine the "sample rate" as the volume (ml) of titrant added per minute from the initial linear portion of the curve.

Calculation