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Carboxypeptidase Y

(Peptidyl-L-amino-acid (-L-proline ) hydrolase; EC

Carboxypeptidase Y (CPY) catalyzes the following reaction:

 Peptidyl-L-amino acid + H2O ------> Peptide + L-amino acid
It is prepared according to the method of Moore & Stein (J. Biol Chem, 211, 907, 1954). It resembles Carboxypeptidase A in its substrate specificity, but it hydrolyzes C-terminal glycine and L-leuicine more rapidly and L-phenylalanine more slowly.

One unit of enzyme activity is defined as that amount of enzyme that catalyzes the hydrolysis of 1 micromole of substrate per minute.

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

  1. 50 mM Sodium phosphate buffer (pH 6.5)
  2. 1 mM Benzyloxycarbonyl-phenylalanyl-L-leucine
  3. Enzyme solution: dilute enzyme with distilled water to a concentration of 1-5 U/ml.

  1. Set the water bath at 25C. Pipette 1.0 ml of substrate solution into a test tube:1mM Benzyloxycarbonlyl-phenylalanyl-L-leucine in 50mM sodium phosphate buffer, pH 6.5.
  2. Incubate at 25C for 15 minutes.
  3. Mix in 50 l of CPY solution diluted to around 0.3 U/ml and let the reaction procede for 15 min.
  4. Stop the reaction by adding1.0 ml of ninhydrin reagent and heat for 20 minutes in boiling water.
  5. Add 5.0 ml of 50% ethanol solution.
  6. Measure (delta)E570 against a blank, zero time sample.

Activity (U/mg) = (ΔE570nm/min)(Total Vol.)(Enz. Diln.)
(Extin. Coeff of ninhydrine)(10 min.)(Enz. Vol.)

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