Carboxypeptidase A (CPA) is an exopeptidase secreted by the pancreas. This enzyme acts preferentially, but not exclusively, on peptide bonds adjacent to the C-terminal aromatic amino acid residues. Peptide bonds involving glycine, aspartic acid and glutamic acid are hydrolyzed slowly while those involving arginine, proline and hydroxyproline are not hydrolyzed.

Carboxypeptidase A possesses a single zinc ion that is tightly coordinated to two histidine imidazole rings and the carboxyl group of a glutamate residue. Bovine CPA has been studied extensively. It has a molecular weight of about 35,000 and the optimum pH for its activity ranges from 7-8.

1. 50 mM Tris/HCl buffer (containing 1.0 M NaCl), pH 7.5.
2. 1 mM Hippuryl-L-phenylalanine in Tris buffer, pH 7.5.
3. 10% Lithium chloride
4. Enzyme solution: dissolve Carboxypeptidase A in 10% Lithium chloride to obtain a concentration of 1-3 U/ml (mg/ml = E278nm X 0.515)

1. Set spectrophotometer (equipped with strip chart recorder and temperature control) at 254 nm and 25°C.
2. Into cuvettes pipette 2.9 ml of Hippuryl-L-phenylalanine. Incubate in spectrophotometer for 5 min. to equilibrate and to establish a blank rate, if any.
3. Add 0.1 ml. of the enzyme solution to the test cuvette, mix, and record the rate of increase in absorbance at 254 nm for 5 min.
4. Calculate the E254nm per minute from the initial part of the curve.