section 3.6
Amino Acid Sequence Determination
47
Peptide
I
NH
I
CH— CH2
c= o
HN:
I
CHR
I
Peptide
Peptide
Methionine residue
NH
CH2— S— CH3
|
CH — CH —
o
JE
h
r
O
JL
1 A
1
A
|
N = C — Br
c = o
N = C
Br
Cyanogen bromide
HN.
|
CHR
Peptide
A CH3— SCN
Methyl thiocyanate
Peptide
Peptide
NH
NH
CH--------CH2
-------------------------------------------
CH--------CH2
C ^
CH2
0
O
NH3
Br-
1
C ^
X H .
I l > ^
■
CHR
HN
Br
Peptidyl homoserine
i
|
lactone
Peptide
CHR
Amino acyl peptide
Peptide
FIGURE 3-12
Cleavage of a peptide chain at methionine residue by cyanogen bromide. The methionine residue is modified to a
carboxy-terminal homoserine lactone residue.
the amino group of an amino acid is reactive, it must be
protected by a blocking group (tertiary butyloxycarbonyl;
t-BOC), so that it does not react with the chloromethyl
groups. The t-BOC is later removed by acid, enabling the
amino group to participate in peptide bond formation. The
degradation products of t-BOC, isobutylene and carbon
dioxide, are removed as gases. The carboxyl group of a
second amino acid, added as a t-BOC derivative, reacts
Peptides obtained from trypsin cleavage:
Ala-Gly-Glu-Lys
Gly-Ala-Met-Arg
lle-Val-Phe
Peptides obtained from cyanogen cleavage:
Ala-Gly-GIu-Lys-Gly-Ala-homoserine lactone*
Arg-lle-Val-Phe
'This residue is derived from methionyl residue.
The complete sequence deduced
from the above overlapping peptides:
Ala-Gly-Glu-Lys-Gly-Ala-Met-Arg-lle-Val-Phe
FIGURE 3-13
Deduction of peptide sequence from analysis of overlapping sequences of
component peptides.
with the amino group of the anchored amino acid in the
presence of the condensing agent, dicyclohexylcarbodi-
imide (DCC). DCC, which removes H20 from the two
functional groups forming a peptide bond, is converted
to dicyclohexylurea. In the next cycle, the t-BOC of a
second amino acid of the solid-phase dipeptide is sim-
ilarly removed, and a third t-BOC-amino acid is added
along with DCC. The stepwise process of peptide syn-
thesis is continued until the desired peptide is formed
(Figure 3-14). The finished peptide can be easily cleaved
from the polystyrene resin without affecting the peptide
linkages.
Advantages of the solid-phase method are amenability
to automation, the almost
1 0 0
% yield of product for
each reaction, the ease of removal of excess reagents
and waste products by washing and filtration of resin
particles, the lack of need for purification of inter-
mediates, and speed. Peptides or proteins that have
been synthesized by the solid-phase method include
ribonuclease, bradykinin, oxytocin, vasopressin, somato-
statin, insulin, and the /1-chain of hemoglobin. The se-
quence analyses for these substances were confirmed by
demonstrating that the synthetic products, constructed on
the basis of sequence data, had the same biological ac-
tivities as those of the corresponding natural substances.
