Aminopeptidases 325
2.3.5. Assay
An assay based on the hydrolysis of glycyl-L-proline at 37°C has
been described. An incubation mixture (0.5 mL) containing 0.1 mL
enzyme preparation in 50 mM glycyl-L-proline, 50 mM Tris-HC1, pH
7.8, and 1 mM MnC12 is prepared and incubated for 1 h at 37°C. The
reaction is then stopped with 1.0 mL of 0.45M TCA, and the quantity
of proline produced measured using the Chinard colorimetric method
(34), with L-proline standards. Chinard's reagent consists of 600 mL
glacial acetic acid, 400 mL orthophosphoric acid (6/14), and 25 g nin-
hydrin, dissolved at 70°C. One milliliter of glacial acetic acid and 1
mL of Chinard's reagent are added to 0.5 mL of reaction mixture. After
10 min at 90°C, absorbance is read at 515 nm. Dilutions should be
made in 0.45M TCA. Using this assay, the enzyme is usually supplied
with a specific activity of 200-300 U/mg of protein (33,35). Measure-
ment of prolidase activity using anAla-Pro substrate, isotachophoresis,
or chromatographic methods has also been described (36,37).2.3.6. Stability
The enzyme is most stable at pH 6-8 in the presence of 0.01M
MnC12. The lyophilized enzyme is stable for many months when stored
at -20°C (38), and is stable for several weeks at 4°C if stored in the
presence of 2 mM MnC12 and 2 mM ~-mercaptoethanol (30).
2.3.7. Activation/Inhibition
Manganous ions are essential for optimal catalytic activity. The
enzyme is inhibited by 4-chloromercuribenzoic acid, iodoacetamide,
EDTA, fluoride, and citrate. However, if Mn 2÷ is added before iodo-
acetamide, no inhibition is observed (31).- Experimental Procedures
- L Removal of Pyroglutamic Acid
from the N-Terminus of a Protein or Peptide
This method is based on the procedure described by Podell and
Abraham (26). The sample (10 mg) is dissolved in 10 mLofdeblocking
buffer (0.1M sodium phosphate buffer, pH 8.0, 5 mM DTT, 10 mM
EDTA, 5% glycerol) and placed in a screw-top vial. Approximately
25 l-tg of enzyme are then added, the vial flushed with nitrogen, and
sealed. Protein and enzyme are allowed to incubate at 4°C for 9 h with