Protein Protocols Protein Protocols

Protein Hydrolysates 565
2. Pronase: The enzyme is stable at 4°C for at least 6 mo and is usually stored as a stock
solution of 5–20 mg/mL in water at –20°C.
3. Aminopeptidase M: The lyophilized enzyme is stable for several years at –20°C. A working
solution can be prepared by dissolving about 0.25 mg of protein in 1 mL of deionized
water to give a solution of approx 6 U of activity/mL. This solution can be aliquoted and
stored frozen for several months at –20°C.
4. Prolidase: The lyophilized enzyme is stable for many months when stored at –20°C
and is stable for several weeks at 4°C if stored in the presence of 2 mM MnCl 2 and 2 mM
β-mercaptoethanol (18).
3. Methods
3.1. Digestion of Proteins (22)
1. Dissolve 0.2-µmol of protein in 0.2 mL of 0.05M ammonium bicarbonate buffer, pH 8.0,
or 0.2 M sodium phosphate, pH 7.0 (see Note 1).
2. Add Pronase to 1% (w/w), and incubate at 37°C for 24 h.
3. Add aminopeptidase M at 4% (w/w), and incubate at 37°C for a further 18 h.
4. Since in many cases the X-Pro- bond is not completely cleaved by these enzymes, to
ensure complete cleavage of proline-containing polypeptides, the aminopeptidase M digest
should be finally treated with 1 µg of prolidase for 2 h at 37°C.
5. The sample can now be lyophilized and is ready for amino acid analysis (see Note 2).
3.2. Digestion of Peptides (22)
This procedure is appropriate for polypeptides less than about 35 residues in length.
For larger polypeptides, use the procedure described in Subheading 3.1.
1. Dissolve the polypeptides (1 nmol) in 24 µL of 0.2 M sodium phosphate buffer, pH 7.0, or
0.05 M ammonium bicarbonate buffer, pH 8.0 (see Note 1).
2. Add 1 µg of aminopeptidase M (1 µL), and incubate at 37°C.
3. For peptides containing 2–10 residues, 8 h are sufficient for complete digestion. For larger
peptides (11–35 residues), a further addition of enzyme after 8 h is needed, followed by a
further 16-h incubation.
4. To ensure complete cleavage at proline residues, finally treat the digest with 1 µg of
prolidase for 2 h at 37°C.
5. The sample can now be lyophilized and is ready for amino acid analysis (see Note 2).
4. Notes
1. Sodium phosphate buffer should be used if ammonia interferes with the amino acid analysis.
2. When using two enzymes or more, there is often an increase in the background amino
acids owing to hydrolysis of each enzyme. It is therefore important to carry out a digestion
blank to correct for these background amino acids.
References
1. Hiramatsu, A. and Ouchi, T. (1963) On the proteolytic enzymes from the commercial protease
preparation of Streptomyces griseus (Pronase P). J. Biochem. (Tokyo) 54(4), 462–464.
2. Narahashi, Y. and Yanagita, M. (1967) Studies on proteolytic enzymes (Pronase) of Streptomyces
griseus K-1. Nature and properties of the proteolytic enzyme system. J. Biochem.
(Tokyo) 62(6), 633–641.
3. Wählby, S. and Engström, L. (1968) Studies on Streptomyces griseus protease. Amino
acid sequence around the reactive serine residue of DFP-sensitive components with esterase
activity. Biochim. Biophys. Acta 151, 402–408.