44
CHAPTER 3
Protein Isolation and Determination of Amino Acid Sequence
HP.
-CH,
H,c.
Dansyl chloride
.CH,
H
o
Cl + H N— C— C — N—
c—
C — N— C— C— N— C— C— O”
I
H
I
H
I
H
I
R,
Rj
Rj
R,
HCI
Base
H O
H O
H
HN— C— C— N— C — C— N-
I
H
I
H
R,
R,
-c—c —N—c—c—O"
I
H
i
R
3
R<
SO
2
H
Acid hydrolysis
Free amino adds
H
H
H
HN— C— COOH++HJN— C— COOH++HJN— C— COOH + +K N — C— COOH
I
I
I
I
R,
Ra
R.
R.
Dansyl derivative
of N-termina! amino
acid (highly fluorescent)
FIG U R E 3-8
Determination of N-terminal amino acid residues by use of dansyl chloride.
Treatment of a peptide with dansyl chloride fol-
lowed by hydrolysis yields a dansyl derivative of the
N-terminal amino acid and other unlabeled amino acids
(Figure 3-8).
The dansyl amino acid is separated and identified by chro-
matographic methods. The dansyl procedure is about 100
times more sensitive than the DNFB method because the
dansyl amino acids are highly fluorescent and therefore
detectable in minute quantities.
In the Edman procedure,
PITC reacts under ba-
sic conditions with the free a-amino group to form
a phenylthiocarbamoyl peptide (Figure 3-9). Treatment
with anhydrous acid yields the labeled terminal amino
residue plus the remainder of the peptide. In this pro-
cess, the terminal amino acid is cyclized to the cor-
responding phenylthiohydantoin derivative (PTH-amino
acid), which can be identified by gas chromatography,
reverse-phase high-pressure liquid chromatography, thin-
layer chromatography, or as the free amino acid after
hydrolysis.
A significant advantage of the Edman procedure is that
on removal of the N-terminal residue, the remaining pep-
tide is left intact and its N-terminal remaining peptide
group is available for another cycle of the procedure. This
procedure can thus be used in a stepwise manner to estab-
lish the sequence of amino acids in a peptide starting from
the N-terminal.
Identification of the C-Terminal Residue
The C-terminal residue is determined by the use of either
a chemical reagent or the enzyme carboxypeptidase. The
