46
chapter 3
Protein Isolation and Determination of Amino Acid Sequence
FIGURE 3-11
Cleavage of a disulfide bond (cystine residue) by performic acid.
the peptide linkage on the C-terminal side of lysine and
arginine residues. Purification of the hydrolysis prod-
ucts is often the most challenging aspect of sequence
determination.
Anion and cation exchange chromatography, paper
chromatography,
and electrophoresis are useful, and
reverse-phase high-pressure liquid chromatography is
used increasingly because of its speed and sensitivity. The
purified peptides are analyzed for amino acid composition
and terminal residues. Small peptides may be sequenced
directly, but large peptides must be further hydrolyzed.
Proteases such as chymotrypsin, pepsin, and papain, which
are much less specific than trypsin, hydrolyze the peptides
formed on tryptic digestion. The amino acid sequences of
the purified peptides may be determined by the sequential
Edman procedure.
Another sequencing technique is indirect analysis fol-
lowing nucleic acid sequencing of a DNA or RNA
fragment corresponding to a specific protein. The uni-
versal genetic code provides information for translating
a nucleic acid sequence into an amino acid sequence.
This method will not correctly identify amino acid se-
quences from proteins that undergo posttranslational mod-
ification or proteins derived from eukaryotic genes with in-
tervening sequences that are not translated (Chapter 25).
However, it is a rapid means of corroborating sequenc-
ing data obtained by the classic slower methods described
above.
TABLE 3-2
Hydrolysis o f Polypeptides at Specific Sites by Selective
Reagents
Cleavage site
H
о
H
R ,
\ /
N-terminal
ч / C\
/ •
••
C-terminal
amino acid
P
N
G
amino acid
residue
/
\
H
Fh
H
О
residue
Amino acid
Amino acid
residue 1
residue 2
(carboxyl side)
(amino side)
Cleavage Reagent
Cleavage Site
Enzymes
Trypsin
Chymotrypsin
Thermolysin
Chemicals
Cyanogen bromide
2-Nitro-5-
thiocyanobenzoate
N-Bromosuccinimide
Hydroxylamine
Mild acid hydrolysis
(70% HCOOH or
0.1 AHC1)
Lys or Arg at amino acid 1
Phe, Trp, or Tyr at amino acid 1
Leu, lie, or Val at amino acid 2
Met at aminio acid 1
Cys at amino acid 2
Tryptophan or tyrosine at
amino acid
1
Rj =Asn; R
2
= Gly
R, =Asp; R2=Pro
Peptide Sequence Confirmation
Once the sequence has been determined, the proper
arrangement of individual peptides in the protein can be
established by identifying the overlapping sequences be-
tween peptides obtained by different cleavage procedures
(Figure 3-13). The ultimate confirmation of sequence de-
termination is protein synthesis. Chemical synthesis of
peptides and proteins of known amino acid sequence
can be accomplished by an elegant, automated, solid-
phase procedure developed by Merrifield et al. Synthesis
begins with the C-terminal amino acid, with each succes-
sive residue added in a stepwise manner. The C-terminal
amino acid is covalently bound to a solid phase by a reac-
tion between the carboxyl group and a chloromethyl group
linked to a phenyl group of the resin polystyrene. Since
