Carboxypeptidase B catalyzes hydrolysis of the basic amino acids lysine, arginine and ornithine from the C-terminal end of polypeptides. The molecular weight is 34,500 daltons, the pH optimum is 7.9, and the pI is 6.0. Carboxypeptidase B is competitively inhibited by arginine, lysine and ornithine. The enzyme is not inhibited by di-isopropylfluorophosphate (DFP), but it is inhibited by metal chelating agents, e.g., 1,10-phenanthroline.
The enzyme is chromatographically purified and supplied as a solution in 100 mM sodium chloride. Chymotrypsin and trypsin 0.02%.
Carboxypeptidase B (CPDB) is a metallocarboxypeptidase that catalyzes the hydrolysis of the basic amino acids, lysine, arginine, and ornithine from the C-terminal position of polypeptides:
Peptidyl-L-lysine(-L-basic amino acid) + H2O --> Peptide + L-lysine(L-basic amino acid)
Carboxypeptidases are secreted as zymogens by the acinar cells of the pancreas. The zymogens are activated by trypsin in the small intestine.
Carboxypeptidase B Assay
Method
Activity is measured by the spectrophotometric method where the reaction velocity is determined by an increase in absorbance at 254 nm resulting from the hydrolysis of hippuryl-L-arginine. One unit causes the hydrolysis of one micromole of hippuryl-L-arginine per minute at 25°C and pH 7.65 under the specified conditions.
Reagents
0.025 M Tris⋅HCl buffer, pH 7.65 containing 0.1 M sodium chloride
0.001 M Hippuryl-L-arginine in 0.025 M Tris⋅HCl pH 7.65 containing 0.1 M sodium chloride
Enzyme
Dilute stock solution with reagent grade water to a concentration of 1-5 units/ml.
Procedure
Set spectrophotometer at 254 nm and 25°C.
Pipette 2.9 ml of substrate into 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 3-4 minutes. Determine ΔA254/minute from the initial linear portion of the curve.
Calculation
where 0.349 is the extinction coefficient of hippuric acid formed during the reaction
C2627056