326 Sweeney and Walkeroccasional mixing. Another 25 ~g of enzyme are then added, and
incubation continued under nitrogen at room temperature for a further
14 h. Since the purpose of deblocking is invariably to render the pro-
tein amenable to sequence analysis, the protein can be desalted either
by dialysis against 0.05M acetic acid and then lyophilized, or by pas-
sage through an HPLC gel-filtration column in 0.1% aqueous TFA.
3.2. N-Terminal Sequence Determination
by Time-Course Hydrolysis
with Aminopeptidase M (39)
Dissolve the polypeptide (1 nmol) in 49 laL of 0.2M sodium phos-
phate buffer, pH 7.0. Add 1 lxL (0.005 U) of aminopeptidase M solu-
tion, and incubate at room temperature. Timed aliquots (5 pL) are then
removed (0, 15, 30, and 45 min, and so on) and analyzed by amino acid
analysis for the presence of free amino acids. The amount of each
amino acid released is plotted against time, and the order of amino
acids deduced from the graph.
3.3. The Use of Aminopeptidase M and Prolidase
to Produce a Peptide/Protein Hydrolysate
for Amino Acid Analysis (40)
Dissolve the polypeptide (1 nmol) in 24 ktl-, of 0.2M sodium phos-
phate buffer, pH 7.0. Add 1 ~tg of aminopeptidase M (1 lxL), and
incubate at 37°C. For peptides containing 2-10 residues, 8 h are suf-
ficient 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. For polypeptides containing more than 35 residues,
digestion with aminopeptidase M alone is insufficient. The polypep-
tide is first digested with the nonspecific protease pronase (1%, v/v),
followed by aminopeptidase M (41,42).
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 treated with 1 ktg
of prolidase for 2 h at 37°C before analysis. The use of two or more
proteolytic enzymes to produce hydrolysis will often lead to an increase
in the level 04' background amino acids. A digestion blank should there-
fore also be analyzed in order to correct for the background amino acids.