Download Amino Acid Analysis

27.8
Introduction to Peptide Structure
Determination
Primary Structure
The primary structure is the amino acid
sequence plus any disulfide links.
Classical Strategy (Sanger)
1. Determine what amino acids are present and
their molar ratios.
2. Cleave the peptide into smaller fragments,
and determine the amino acid composition of
these smaller fragments.
3. Identify the N-terminus and C-terminus in the
parent peptide and in each fragment.
4. Organize the information so that the
sequences of small fragments can be
overlapped to reveal the full sequence.
27.9
Amino Acid Analysis
Amino Acid Analysis
Acid-hydrolysis of the peptide (6 M HCl, 24 hr)
gives a mixture of amino acids.
The mixture is separated by ion-exchange
chromatography, which depends on the
differences in pI among the various amino
acids.
Amino acids are detected using ninhydrin.
Automated method; requires only 10-5 to 10-7 g
of peptide.
27.10
Partial Hydrolysis of Proteins
Partial Hydrolysis of Peptides and Proteins
Acid-hydrolysis of the peptide cleaves all of the
peptide bonds.
Cleaving some, but not all, of the peptide bonds
gives smaller fragments.
These smaller fragments are then separated
and the amino acids present in each fragment
determined.
Enzyme-catalyzed cleavage is the preferred
method for partial hydrolysis.
Carboxypeptidase
Carboxypeptidase is a proteolytic enzyme
(catalyzes the hydrolysis of proteins).
O
O
+
H3NCHC
R
protein
C
O
–
NHCHCO
R
Carboxypeptidase
Carboxypeptidase is a proteolytic enzyme
(catalyzes the hydrolysis of proteins).
Carboxypeptidase is selective for cleaving
the peptide bond to the C-terminal amino acid.
O
O
+
H3NCHC
R
protein
C
O
–
NHCHCO
R
Trypsin
Trypsin is selective for cleaving the peptide bond
to the carboxyl group of lysine or arginine.
O
O
O
NHCHC
NHCHC
NHCHC
R
R'
R"
lysine or arginine
Chymotrypsin
Chymotrypsin is selective for cleaving the peptide
bond to the carboxyl group of amino acids with
an aromatic side chain.
O
O
O
NHCHC
NHCHC
NHCHC
R
R'
R"
phenylalanine, tyrosine, tryptophan
27.11
End Group Analysis
End Group Analysis
Amino sequence is ambiguous unless we know
whether to read it left-to-right or right-to-left.
We need to know what the N-terminal and Cterminal amino acids are.
The C-terminal amino acid can be determined
by carboxypeptidase-catalyzed hydrolysis.
Several chemical methods have been
developed for identifying the N-terminus. They
depend on the fact that the amino N at the
terminus is more nucleophilic than any of the
amide nitrogens.
Sanger's Method
The key reagent in Sanger's method for
identifying the N-terminus is 1-fluoro-2,4dinitrobenzene.
1-Fluoro-2,4-dinitrobenzene is very reactive
toward nucleophilic aromatic substitution
(Section 23.5).
NO2
O2N
F
Sanger's Method
1-Fluoro-2,4-dinitrobenzene reacts with the
amino nitrogen of the N-terminal amino acid.
NO2
O2N
O
F + H2NCHC
O
NHCHC
O
NHCH2C
CH(CH3)2 CH2C6H5
O
–
NHCHCO
CH3
Sanger's Method
1-Fluoro-2,4-dinitrobenzene reacts with the
amino nitrogen of the N-terminal amino acid.
NO2
O2N
O
O
F + H2NCHC
NHCHC
NHCH2C
O2N
O
NHCHC
O
NHCHC
O
NHCH2C
CH(CH3)2 CH2C6H5
–
NHCHCO
CH3
CH(CH3)2 CH2C6H5
NO2
O
O
O
–
NHCHCO
CH3
Sanger's Method
Acid hydrolysis cleaves all of the peptide bonds
leaving a mixture of amino acids, only one of
which (the N-terminus) bears a 2,4-DNP group.
NO2
O2N
O
NHCHC
O
NHCHC
O
NHCH2C
CH(CH3)2 CH2C6H5
O
–
NHCHCO
CH3
Sanger's Method
Acid hydrolysis cleaves all of the peptide bonds
leaving a mixture of amino acids, only one of
which (the N-terminus) bears a 2,4-DNP group.
NO2
O2N
O
NHCHCOH
CH(CH3)2
H3O+
NO2
O2N
O
NHCHC
O
NHCHC
O
NHCH2C
CH(CH3)2 CH2C6H5
O
–
NHCHCO
CH3
Sanger's Method
Acid hydrolysis cleaves all of the peptide bonds
leaving a mixture of amino acids, only one of
which (the N-terminus) bears a 2,4-DNP group.
NO2
O
O
O
O
+
+
+
NHCHCOH + H3NCHCO– + H3NCH2CO– + H3NCHCO–
O2N
CH(CH3)2
CH3
CH2C6H5
H3O+
NO2
O2N
O
NHCHC
O
NHCHC
O
NHCH2C
CH(CH3)2 CH2C6H5
O
–
NHCHCO
CH3
27.12
Insulin
Insulin
Insulin is a polypeptide with 51 amino acids.
It has two chains, called the A chain (21 amino
acids) and the B chain (30 amino acids).
The following describes how the amino acid
sequence of the B chain was determined.
The B Chain of Bovine Insulin
Phenylalanine (F) is the N terminus.
Pepsin-catalyzed hydrolysis gave the four peptides:
FVNQHLCGSHL
VGAL
VCGERGF
YTPKA
The B Chain of Bovine Insulin
FVNQHLCGSHL
VGAL
VCGERGF
YTPKA
The B Chain of Bovine Insulin
Phenylalanine (F) is the N terminus.
Pepsin-catalyzed hydrolysis gave the four peptides:
FVNQHLCGSHL
VGAL
VCGERGF
YTPKA
Overlaps between the above peptide sequences were
found in four additional peptides:
SHLV
LVGA
ALT
TLVC
The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALY
YLVC
VCGERGF
YTPKA
The B Chain of Bovine Insulin
Phenylalanine (F) is the N terminus.
Pepsin-catalyzed hydrolysis gave the four peptides:
FVNQHLCGSHL
VGAL
VCGERGF
YTPKA
Overlaps between the above peptide sequences were
found in four additional peptides:
SHLV
LVGA
ALT
TLVC
Trypsin-catalyzed hydrolysis gave GFFYTPK which
completes the sequence.
The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALY
YLVC
VCGERGF
GFFYTPK
YTPKA
The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALY
YLVC
VCGERGF
GFFYTPK
YTPKA
FVNQHLCGSHLVGALYLVCGERGFFYTPKA
Insulin
The sequence of the A chain was determined
using the same strategy.
Establishing the disulfide links between cysteine
residues completed the primary structure.
Primary Structure of Bovine Insulin
N terminus
of A chain
S
S
C terminus
of A chain
15
5
E Q C
V
C S L Y Q L
I
F
E N
20
C
V
YC
A S
S
10
N
S
S
H
L
N Q
V
S
C
F
G S H L V G A L Y L V
5
C
15
G 20
10
N terminus
E
of B chain
G R
F
F
Y
K P T
A
C terminus
25
30
of B chain
27.13
The Edman Degradation and
Automated Sequencing of
Peptides
Edman Degradation
1. Method for determining N-terminal amino
acid.
2. Can be done sequentially one residue at a
time on the same sample. Usually one can
determine the first 20 or so amino acids from
the N-terminus by this method.
3. 10-10 g of sample is sufficient.
4. Has been automated.
Edman Degradation
The key reagent in the Edman degradation is
phenyl isothiocyanate.
N
C
S
Edman Degradation
Phenyl isothiocyanate reacts with the amino
nitrogen of the N-terminal amino acid.
O
C6H5N
C
S
+
+ H3NCHC
R
NH
peptide
Edman Degradation
S
O
C6H5NHCNHCHC
peptide
NH
R
O
C6H5N
C
S
+
+ H3NCHC
R
NH
peptide
Edman Degradation
S
O
C6H5NHCNHCHC
NH
peptide
R
The product is a phenylthiocarbamoyl (PTC)
derivative.
The PTC derivative is then treated with HCl in
an anhydrous solvent. The N-terminal amino
acid is cleaved from the remainder of the
peptide.
Edman Degradation
S
O
C6H5NHCNHCHC
peptide
NH
R
HCl
S
C6H5NH
C
C
N
CH
R
O
+
+
H3N
peptide
Edman Degradation
The product is a thiazolone. Under the
conditions of its formation, the thiazolone
rearranges to a phenylthiohydantoin (PTH)
derivative.
S
C6H5NH
C
C
N
CH
R
O
+
+
H3N
peptide
Edman Degradation
C6H5
S
N
C
C
The PTH derivative is
isolated and identified.
The remainder of the
peptide is subjected to
a second Edman
degradation.
O
CH
HN
R
S
C6H5NH
C
C
N
CH
R
O
+
+
H3N
peptide