Enzymatic Assay of Carboxypeptidase A

1. Objective

To standardize a procedure for the assay of Carboxypeptidase A.

2. Scope

This procedure applies to all products that have a specification for carboxypeptidase A including, but not limited to, carboxypeptidase A from bovine pancreas (Product No. C9268).

3. Definitions

3.1 Purified Water = water from a deionizing system, resistivity > or = 18MΩ•cm @ 25 ºC

3.2 Carboxypeptidase A Unit Definition – One unit will hydrolyze 1.0 μmol of hippuryl-L-phenylalanine per minute at pH 7.5 at 25 ºC.

4. Discussion

Hippuryl - L- Phenylalanine ^{Carboxypeptidase A} > Hippuric acid + Phenylalanine

5. Responsibilities

Analytical services laboratory personnel should follow this procedure as written.

6. Safety

Refer to Safety Data Sheets (SDS) for hazards and appropriate handling precautions.

7. Procedure

7.1 CONDITIONS:
T = 25 °C, pH = 7.5, A_254nm, Light path = 1 cm

7.2 METHOD:
Continuous Spectrophotometric Rate Determination

7.3 REAGENTS:

7.3.1 25 mM Tris HCl buffer with 500 mM sodium chloride, pH 7.5 at 25 °C (Buffer)
Prepare a solution in purified water containing 3.03 mg/mL Trizma Base, such as Product No. T1503, and 29.2 mg/mL sodium chloride, such as Product No. S9888. Adjust the pH to 7.50 at 25 °C using 1 N hydrochloric acid. Keep solution at room temperature.

7.3.2 1.0 mM Hippuryl-L-Phenylalanine Solution (Hippuryl-L-Phe)
Prepare a solution in 200 Proof Ethanol containing 32.6 mg/mL hippuryl-L-phenylalanine, such as Product No. H6875. Once the sample is completely in solution in ethanol, further dilute the solution in Reagent 7.3.1 (Buffer) to 0.326 mg/mL. Do not adjust the pH of this solution. Keep the solution at room temperature and use within three hours of preparation.

7.3.3 1.0 M Sodium Chloride Solution (Enzyme Diluent)
Prepare a solution in purified water containing 58.4 mg/mL Sodium Chloride, such as Product No. S9888. Keep solution at room temperature.

7.3.4 Carboxypeptidase A Enzyme Solution (Enzyme)

7.3.4.1 Before use, prepare a solution in room temperature Reagent 7.3.3 (Enzyme Diluent) containing 4-8 units/mL of Carboxypeptidase A.

7.3.4.2 Do not dilute the enzyme in cold Reagent 7.3.3.

7.3.4.3 Make a fresh dilution of enzyme in reagent 7.3.3 (Enzyme Diluent) for each aliquot.

7.3.4.4 Perform independent dilutions for each sample and repeat to verify consistency of aliquots taken from the retainer or bulk.

7.3.4.5 Prior to pulling a sample, be sure to shake the sample to insure all material that has settled to the bottom is in a homogeneous solution.

7.4 PROCEDURE

7.4.1 Run one aliquot at a time because the maximum rate is typically in the first minute.

7.4.2 Blank the spectrophotometer against reagent 7.3.2. Because of the high absorbency of reagent 7.3.2, the assay needs to be run on instrument that is accurate above an absorbency of 2.0.

7.4.3 Pipette the following (in milliliters) into suitable quartz cuvettes:

	Test	Blank
Hippuryl-L-Phe (Reagent 7.3.2)	2.90	2.90

7.4.4 Equilibrate to 25 °C using a suitably thermostatted spectrophotometer. Then add (in milliliters):

Enzyme Diluent (Reagent 7.3.3)	----	0.10
Enzyme (Reagent 7.3.4)	0.10	----

7.4.5 Mix by inversion and record the increase in absorbance at 254nm for approximately 5 minutes. Obtain the fastest linear rate (ΔA_254nm/minute) over a one minute interval for the test and the blank reactions. The ΔA_254nm/minute must be from 0.05 to 0.1 for each reaction for data to be valid.

7.5 CALCULATIONS

7.5.1	Units/mL enzyme =	(ΔA254nm/min Test - ΔA254nm/min Blank)(3.00)(df)
		(0.36)(0.10)

7.5.2	Units/mg solid =	Units/mL enzyme
		mg solid/mL enzyme

7.5.3	Units/mg protein =	Units/mL enzyme
		mg protein/mL enzyme

3.00 = Total volume (in milliliters) of assay
df = Dilution Factor
0.36 = Millimolar Extinction Coefficient of hippuric acid at 254 nm
0.10 = Volume (in milliliters) of enzyme used

7.6 FINAL ASSAY CONCENTRATION :
In a 3.00 mL reaction mix, the final concentrations are 24 mM Tris, 0.96% ethanol, 0.97 mM hippuryl-L-phenylalanine, 512 mM sodium chloride, and 0.4-0.8 units carboxypeptidase A.

8. Reference

8.1 Bergmeyer, H.U., Gawehn, K., and Grassl, M. (1974) Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.) Volume 1, 2^nd ed., 436-437, Academic Press, Inc. New York, NY