section 3.6
Amino Acid Sequence Determination
43
elution times for each amino acid. The amino acids are
differentially eluted by the use of a stepwise pH gradient
varying between pH 3 and pH 5 (Figure 3-6). As each
amino acid elutes, it is reacted with ninhydrin at 100°C
to produce a deep blue or purple color (yellow for pro-
line and hydroxyproline). The color intensity is measured
spectrophotometrically to quantitate the amino acids.
The process of separation and quantitation of amino
acids has been automated. In one automated method, a
single cation exchange resin column separates all the
amino acids in the protein hydrolysate. The analyzer is
capable of detecting as little as
1 - 2
nmol of an amino
acid and a complete analysis can be obtained in about
4 hours. In newer procedures, the complete analysis can
be performed in about lhour and permit detection of as
little as 1-2 nmol of an amino acid. Picomole amounts
of amino acids can be determined when the separated
amino acids are coupled to fluorescent reagents such as
o-phthalaldehyde. Amino acid separation and quantitation
can also be accomplished by reverse-phase high-pressure
liquid chromatography of amino acid derivatives—a rapid
and sensitive procedure.
3.6 Amino Acid Sequence Determination
Determination of the amino acid sequence of a protein
involves the following steps:
1. Identification of the N- and C-terminal amino acid
residues,
2. Cleavage of any disulfide bonds present,
3. Limited cleavage of the peptide into overlapping
smaller fragments,
4. Purification of the fragments, and
5. Their stepwise cleavage into individual amino acid
residues.
Identification of the N-Terminal Residue
Determination of the N-terminal residue is carried out
by
labeling
the free unprotonated a-amino groups.
Three
alternative
labeling
reagents
are
used:
2,4-
dinitrofluorobenzene (DNFB; Sanger’s reagent), dansyl
chloride (l-dimethylaminonaphthalene-5-sulfonyl chlo-
ride), and phenylisothiocyanate (PITC; Edman’s reagent).
DNFB and dansyl chloride react with free amino groups
under basic conditions.
The labeled peptide is hy-
drolyzed with acid to yield the labeled N-terminal residue
and other free amino acids (Figure 3-7). The 2,4-
dinitrophenyl amino acid derivatives (DNP-amino acids)
have a yellow color and are separable by chromatographic
methods and identifiable by comparison with reference
DNP-amino acids. DNFB reacts with the e-amino groups
of lysyl residues to yield e-DNP-lysine after hydrolysis.
N-Terminal lysine produces
a,
e-di(DNP)-lysine, whereas
an internal lysine produces a derivative with only one dini-
trophenyl group (e-DNP-lysine).
H
o
H
o
H
o
2,4 - Dinitroflouro-
benzene
(DNFB)
H2N— C— C— N — C— C— N— Ç— C-
I
H
I
H
I
R,
R*
R3
Tetrapeptide
HF-
Base
H
1
0
II
H
O
1
II
H O
H
i
1
11
1
1— c-
- c -
-N-— c — c - - N -
1
0
1
0
1
Z
1
0
1
1
1
R,
H
1
R2
H
1
H
1
Rj
R„
H
O
I
II
_
-c —c — o
I
R.
o
Acid hydrolysis
Free amino acids
H
O
H
0
H
1
O
H
1
1— c -
1
1
II
-C — OH +
+H,N— C—
3
I
II
-c—
OH ■*
+
1
•
H3N — C -
1
o =
1
0
X
+
y
z
1
- c —1
I
1
1
R,
1
r 2
R3
1
R„
O
-OH
FIGURE 3-7
Determination of N-terminal amino acid residues by use of 2,4-dinitrofluorobenzene (Sanger’s reagent).
