That amount of enzyme which hydrolyzes 1 micromole of hippuryl-L-arginine per minute at 25°C and pH 7.65.
Assay Method
Carboxypeptidase B activity is determined from the increase in absorbance due to the hydrolysis of hippuryl-L-arginine at 254 nm.
Applications
Carboxypeptidase B, (CPB) (EC 3.4.12.3) 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.
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.
Reagents
25 mM Tris/HCl buffer, pH 7.65 (containing 0.1 M NaCl).
1 mM Hippuryl-L-arginine in Tris buffer, pH 7.65.
Enzyme solution: dilute enzyme with double distilled water to a concentration of 1-5 U/ml.
(mg/ml = E278nm x 0.476).
Procedure
Set spectrophotometer (equipped with strip chart recorder and temperature control) at 254 nm and 25°C.
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.
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.
Calculate the (delta)E254nm per minute from the initial linear portion of the curve.
