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Carboxypeptidase B

(Peptidyl-L-lysine [L-arginine] hydrolyase; EC

Carboxypeptidase B, (CPB) like carboxypeptidase A, is a pancreatic exopeptidase. Unlike carboxypeptidase A, however, carboxypeptidase B catalyzes the hydrolysis of the peptide bonds involving basic amino acids lysine, arginine and ornithine. This hydrolysis occurs at the C-terminal bond in these polypeptides. Following is a representative reaction showing the catalytic activity of carboxypeptidase B:

Peptidyl-L-lysine + H2O ------> Peptide + L-Lysine
Carboxypeptidase B shows minimal activity towards Carboxypeptidase A substrates. It is a metalloenzyme containing zinc. Porcine carboxypeptidase B has a molecular weight of 34,300 (Folk, J. E., Piez, K.A., Carroll, W.R., and J. Gladner, J. Biol Chem, 235, 2272, 1960).

1.Source: Porcine Pancreas
   Form: Freeze-dried powder 
   Solubility: Distilled water or dilute buffer 
   Stability: Store at -20 C (-4 F) 
   Activity (approx.): 50 U/mg protein 
   Protein (approx.): 80% (biuret) 
   Catalog No.: 121A0200 
2.Source: Porcine Pancreas
   Form: Frozen solid in NaCl 
   Solubility: Distilled water or dilute buffer 
   Stability: Store at -20 C (-4 F) 
   Activity (approx.): 170 U/mg protein 
   Protein (approx.): 10-20 mg/ml (biuret) 
   Catalog No.: 121H0200 

That amount of enzyme which hydrolyzes 1 micromole of hippuryl-L-arginine per minute at 25C and pH 7.65.

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

  1. 25 mM Tris/HCl buffer, pH 7.65 (containing 0.1 M NaCl).
  2. 1 mM Hippuryl-L-arginine in Tris buffer, pH 7.65.
  3. Enzyme solution: dilute enzyme with double distilled water to a concentration of 1-5 U/ml.

    (mg/ml = E278nm x 0.476).

  1. Set spectrophotometer (equipped with strip chart recorder and temperature control) at 254 nm and 25C.
  2. Into quartz cuvettes pipette 2.9 ml Hippuryl-L-arginine substrate. 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 absorbance at 254 nm for 5 min.
  4. Calculate the (delta)E254nm per minute from the initial linear portion of the curve.

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

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