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Carboxypeptidase A

(Peptidyl-L-amino acid hydrolase; EC

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.

That amount of enzyme which hydrolyzes 1 micromole of hippuryl-L-phenylalanine per minute at 25C and pH 7.5.

Carboxypeptidase A activity is determined from the increase in absorbance at 254 nm, due to the hydrolysis of hippuryl-L-phenylalanine.

  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 25C.
  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 (delta)E254nm per minute from the initial part of the curve.

Activity (U/mg) = (ΔE254nm/min)(Total Vol.)(Enz. Diln.)
(0.36)(mg Enz./ml)

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