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