section
3.6
Amino Acid Sequence Determination
45
Phenylisothiocyanate
(PITC)
Peptide
R,
Phenylthiohydantoin
derivative of N-terminal
amino acid (PTH-amino acid)
FIGURE 3-9
Determination of the N-terminal residue by the Edman procedure. After
removal of the N-terminal amino acid, the remainder of the peptide
remains intact and a new N-terminal amino acid is available for removal by
the next reaction cycle.
chemical reagent hydrazine forms aminoacyl hydrazides
with every residue
except
the C terminus (Figure 3-10).
The C terminus is thus readily identified by chromato-
graphic procedures. The disadvantage of hydrazinolysis
is that the entire sample is used to determine just one
residue.
Carboxypeptidase is an exopeptidase that specifically
hydrolyzes the C-terminal peptide bond and releases
the C-terminal amino acid. Two problems are associ-
ated with its use: the substrate specificity of the en-
zyme and the continuous action of the enzyme. The
continuous action may yield the second, third, and ad-
ditional residues from some chains even before the ter-
minal residues on every chain are quantitatively released.
Thus, it may be difficult to determine which residue is
the C terminus. However, monitoring the sequential re-
lease of amino acids can often reveal the sequence of
several residues at the C terminus. Concerning speci-
ficity, carboxypeptidase A releases all C-terminal residues
except Lys, Arg, and Pro; carboxypeptidase B cleaves
C-terminal Arg and Lys residues; and carboxypeptidase
C hydrolyzes C-terminal Pro residues. Thus, more than
one method may be needed to establish the C-terminal
amino acid.
Selective Hydrolysis Methods
Cleavage of disulfide bonds occurs before hydrolysis of
the protein into peptides. Disulfide bonds may be cleaved
oxidatively, or they may be reduced and alkylated. Treat-
ment of the native protein with performic acid, a powerful
oxidizing agent, breaks disulfide bonds and converts cys-
tine residues to cysteic acid (Figure 3-11). Reduction of
the disulfide linkage by thiols, such as /3-mercaptoethanol,
yields reactive sulfhydryl groups. These groups may be
stabilized by alkylation with iodoacetate or ethyleneimine
to yield the carboxymethyl or aminoethyl derivative,
respectively.
Hydrolysis of a protein into peptides can be ac-
complished by group-specific chemical and enzymatic
reagents (Table 3-2). N-Bromosuccinimide and cyanogen
bromide hydrolyze proteins at tryptophan and methionine
(Figure 3-12) residues, respectively. Trypsin hydrolyzes
H O
H O
H O
H O
t
I
II
I
II
I
II
I
II
_
+H3N— C— c — N— C— c — N— c — c — N— C— c — O
I
H
i
H
I
H
I
R,
R,
R3
R4
NH2NH2 (hydrazine)
H
0
H
o
h
0
H
0
1
II
+
1
II
1
II
+
1
II
Ç—
R,
-C— NHNH3 + H3N— C—
1
R,
-c — nhnh2 + H,N— C—
R3
-c — nhnh2 + H3N— C—
1
R.
-c —
Aminoacyl hydrazides
C- terminal
amino acid
F IG U R E 3-10
Determination of C-terminal amino acid residues by use of hydrazine.
