Carboxypeptidase Y 315
2.3. Physical and Chemical Properties
CPY has a mol mass of 61,000-63,000 Da as determined by SDS gel
electrophoresis (3,11). It is a glycoprotein, having a single polypeptide
chain of about 430 residues with 16 residues of glucosamine in the
carbohydrate moiety. About 96%, i.e., 416 of approx 430 amino acid
residues, have been sequenced with tentative assignment of the carbo-
hydrate attachment sites, and the molecule shown to contain one sulf-
hydryl group, either four or five disulfide bridges, and five methionine
residues (12,13).
2.4. pH Optimum
The optimum pH for hydrolysis of acidic amino acids is pH 5.5,
while that for neutral and basic amino acids is pH 7 (5,14).
2.5. Assay
The substrate N-carbobenzoxy-Phe-Ala (N-CBZ-Phe-Ala) is pre-
pared as a 100-mM stock solution in methanol and diluted when required
in 50 mM MES buffer, pH 6.75, to give a substrate solution that is 2
mM in N-CBZ-Phe-Ala and contains 2% methanol. Ten micrograms
of enzyme are added to 3 mL of substrate solution, and the decrease
in absorbance at 230 nm measured with time. One unit is defined as the
amount of enzyme that will hydrolyze 1.0 ~tlol of N-CBZ-Phe-AIa to
N-CBZ-L-phenylalanine and L-alanine/min at pH 6.75 at 25°C (EM230
= 191.5). Using this assay, the enzyme is normally provided with a
specific activity of 100-130 U/mg of protein. Esterase and anilidase
assays have also been well documented (10).
2.6. Stability
The lyophilized enzyme is stable for 6-12 mo if stored at 4°C. A
suspension of the enzyme in saturated ammonium sulfate can be stored
at -20°C indefinitely. When dissolved in water and dialyzed against
water to give an approx 1% solution of the enzyme, this solution can
be aliquoted and stored at -20°C for at least 2 yr. Diluted solutions
(<0.1 mg/mL) lose activity fairly quickly and should therefore be
prepared just before use (5). The enzyme is fully stable in the presence
of 10% methanol at pH 5.5-8.0 for 8 h at 25°C, and in 20% methanol,
80% activity remains after incubation at pH 7 for 24 h (15). Enzyme
activity is rapidly lost below pH 3 (3) or above 60°C (11).