Clostridiopeptidase A Assay

Method: The assay is based on the enzyme-specific splitting of a Pz-Pro-Leu-Gly-Pro-Arg substrate between leucine and glycine (Wünch and Heidrich, 1963). This results in a colored lipophilic fragment and a non-colored tripeptide. The change in absorbance of the fragment at 320 nm per unit time is a measure of enzyme activity.

Reagents

• 0.1 M Tris≥HCl, pH 7.1
• 0.1 M Calcium chloride
• 25 mM Citric acid, pH 3.5
• Ethyl acetate
• Anhydrous sodium sulfate
• Substrate: Pz-Pro-Leu-Gly-Pro-Arg (molecular weight 812.93). Dissolve to 5 mg/ml in methanol. Dilute to 1 mg/ml with Tris buffer.
• Standard: Pz-Pro-Leu (molecular weight 466.54). Dissolve to 5 mg/ml in methanol. Dilute to 1 mg/ml with Tris buffer.

Enzyme

Stock solution: Dissolve at 1 mg/ml in reagent grade water. Dilute stock 1:20 and 1:50 for assay.

Procedure

Standard Curve

	Micrograms of Standard
	0	50	100	200	400	600	800
Standard	0ml	.05ml	.1ml	.2ml	.4ml	.6ml	.8ml
Tris buffer	1.1ml	1.05ml	1.0ml	.9ml	.7ml	.5ml	.3ml
Calcium chloride	.2ml	.2ml	.2ml	.2ml	.2ml	.2ml	.2ml

Mix well and transfer 0.5 ml to labeled tubes containing 1 ml citric acid and 5 ml ethyl acetate. Vortex for 15 seconds. All the phases separate at room temperature. Transfer the organic phase to tubes containing 350 mg sodium sulfate. Shake gently to allow ethyl acetate to dry. Read the A₃₂₀ of the dried ethyl acetate vs. air.

Determine the net A₃₂₀ (test - blank) and plot A₃₂₀ nm/≥g standard from the slope of the curve. Determine the calibration factor as follows:

Enzyme Assay: Pipette 0.2 ml calcium chloride and 1.0 ml substrate into a series of numbered tubes. Incubate in a water bath at 25°C to achieve temperature equilibration. At timed intervals, add 0.1 ml enzyme dilution to the respective tubes. Include 2 tubes with 0.1 ml water as blanks. Incubate exactly 15 minutes at 25°C and at timed intervals withdraw 0.5 ml and transfer to tubes containing 1 ml citric acid and 5 ml ethyl acetate. Proceed as with standard curve, reading A₃₂₀ of dried ethyl acetate vs. air. Determine net A₃₂₀.

Calculation