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Carboxypeptidase A - Assay

Source:
Bovine Pancreas
CAS:
11075-17-5
EC:
3.4.17.1
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Carboxypeptidase A Assay

Method
The determination of reaction velocity is based upon the method of Folk and Schirmer (1963). The rate of hydrolysis of hippuryl-L-phenylalanine is determined by measuring the increase in absorbance at 254 nm. One unit hydrolyzes one micromole of hippuryl-L-phenylalanine per minute at pH 7.5 and 25°C under the specified conditions.
Reagents
  • 0.025 M Tris⋅HCl buffer containing 0.5 M sodium chloride, pH 7.5
  • 0.001 M Hippuryl-L-phenylalanine in 0.025 M Tris⋅HCl, pH 7.5 with 0.5 M sodium chloride
  • 10% Lithium chloride
Enzyme

Dissolve in 10% lithium chloride to a concentration of 1-3 units per ml. The enzyme crystals are not readily soluble in the diluent. Do not use solution for assay until the solution has cleared.

Read A278 in cold 10% lithium chloride.

equation

Procedure

Adjust spectrophotometer to 254 nm and 25°C.

Pipette 2.0 ml of substrate into each cuvette and incubate in spectrophotometer at 25°C for 3-4 minutes to reach temperature equilibration and establish blank rate, if any. Add 0.1 ml of diluted enzyme and record increase in A254 for 3-5 minutes. Determine ΔA254 / minute from the initial linear portion of the curve.

Calculation

where 0.36 is the extinction coefficient of hippuric acid formed during the reaction

Carboxypeptidase A Products

Description
Activity
Code
Cat. #
Size
Price
Description
Carboxypeptidase A, PMSF Treated
Source:
Bovine Pancreas
2X Crystallized. PMSF treated to inhibit tryptic and chymotryptic activities. Carboxypeptidase B, trypsin, and chymotrypsin less than 2.0%, 0.02% and 0.02% respectively. An aqueous suspension with toluene added as a preservative.
Store at 2-8°C.
…
Activity
≥35 units per mg protein
Code
COAPMS