Post on 01-Jul-2021
Peptide Nomenclature and Structure Determination
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PeptidesIntroduction
• A peptide is any polymer of amino acids linked by amide
bonds between the amino group of one amino acid and the
carboxyl group of the neighbouring amino acid.
• Each amino acid unit in the peptide is called a residue
(amino acid residue).
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• The term “peptides” denotes relatively small compounds,
which resemble proteins except that they are composed of
fewer than 50 amino acid residues.
• Polypeptides of more than 50 amino residues are called
proteins.
H2N
R
COOH
H
H2N
R'
COOH
H
+ H2N
R
H
C
O
NH
R'
COOH
HLoss of H2O
Peptide bond
C-terminal endN-terminal end
Peptide Nomenclature• By convention, a peptide is written with the amino acid
residue having a free amino group (NH3+) on the left and the
amino acid residue with a free carboxyl group (CO2-) on the
right.
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• The end of the peptide with the free amino group is called the
N-terminal or the N-terminus and the end with the free
carboxyl group is called the C-terminal or C-terminus.
• Thus, peptide structures are drawn with the N-terminus at the
left and the C-terminus at the right.
CH2N
R
H
C
O
CNH
R'
COOH
H
Peptide bond
C-terminal endN-terminal end
Peptide Nomenclature• As in the naming of amino acids, where the carboxylic acid
group takes precedence over the amino group, in peptides,
the amino acid with the free carboxylic acid group provides
the root name of the peptide.
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Peptide Nomenclature
• Peptides are named beginning at the N-terminus, and the
names of amino acid residues (all except the last) are
derived by dropping the suffix –ine in the name of the
respective amino acids and replacing them with the –yl suffix
of the acyl groups.
• The N-terminus and intermediate amino acids of a peptide
are named as acyl derivatives of the C-terminal amino acid.
For example, the two possible dipeptides from condensation
of glycine and alanine have the following names:
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CH3N
H
H
C
O
CNH
CH3
C
H
Peptide bond
C-terminal endN-terminal end
CH3N
CH3
H
C
O
CNH
H
C
H
Peptide bond
C-terminal endN-terminal end
Glycylalanine (Gly-Ala)
Alanylglycine (Ala-Gly)
O
O
O
O
Peptide StructureSequence of a Peptide
• The precise order of bonding of amino acids from the N-
terminus to the C-terminus in a peptide is called its amino
acid sequence.
• The sequence of a peptide, thus, denotes the order of
arrangement of amino acid residues in a peptide.
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CH3N
H
H
C
O
CNH
CH3
H
Peptide bond
C-terminal endN-terminal end
Glycylalanylphenylalanine
O
O
NHC
O
C C
Three-letter name: Gly-Ala-Phe
H
Ph
There are six possible tripeptides that can be derived from the aminoacids (glycine, alanine and phenylalanine)
Gly-Ala-PheGly-Phe-AlaAla-Gly-PheAla-Phe-GlyPhe-Gly-AlaPhe-Ala-Gly
Sequence of a Peptide
• An accurate description of the structure of any peptide must
specify its amino acid sequence, since its biochemical
properties depends on this amino acid sequence.
• Bradykinin, a nonapeptide present in blood plasma, is
involved in regulating blood pressure.
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H3N CH
NH
NH2HN
NC
O
CH NC
O
CHHNC
O
CH2
HNC
O
CH
CH2
HNC
O
CH
CH2
OH
NC
O
CHHNC
O
CH
CH2
HNC
O
CH
NH
OC
O
NH2HN
N-terminus C-terminus
Arginine Proline Proline Glycine Phenylalanine Serine Proline Phenylalanine Arginine
• The name of bradykinin: is
arginylprolylprolylglycylphenylalanylserylprolylphenylalanylargi
nine.
• The shorthand system that uses the three-letter codes of he
amino acids is more convenient. Bradykinin has the
abbreviated name: Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg.
Levels of Peptide Structure
There are several levels of peptide structure:
• The primary structure of a peptide consists of the covalently
bonded structure of a peptide. This comprises of the
peptide’s composition (how many and which amino acids are
present) and their amino acid sequence in the peptide chain.
• The secondary, tertiary and quaternary structures refer to the
three-dimensional aspects of peptide structure.
• The three-dimensional structure of a peptide includes the
conformatons in which the peptide chain is folded and any
hydrogen-bonding interactions between the component
amino acid residues.
• The three-dimensional structure of the peptide significantly
influences the biological activity of the peptide.
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Primary Structure of a Peptide Determining the Primary Structure of a Peptide
• Determining the primary structure of a peptide can be truly a
formidable task, since the 20 standard amino acids provide a
number of molecular building blocks large enough to produce
a staggering number of sequences and sizes of peptides.
• To determine the primary structure of a particular peptide,
one must know:
i. The composition of the peptide i.e what amino acid
residues make up the peptide and how many of each
there are, and
ii. The sequence of the peptide i.e the order in which the
amino acid residues are arranged in the peptide.99:48 AM
The Composition of a Peptide:Hydrolysis of a Peptide
• The first step in determining the structure of a peptide is to
identify the composition of the peptide. This is accomplished
by subjecting the peptide to acid-catalysed hydrolysis by
heating at 110 oC in 6 M hydrochloric acid for about 24 h.
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CH3N C
R
H O
CHN C
R'
H O
CHN C
R''
H
O
O
Hydrolysis of Peptide
6M HCl, 110 oC
24 hours
CH3N C
R
H O
OH CH3N C
R''
H
OH
O
Cl Cl+ +CH3N C
R'
H O
OHCl
• Under these conditions all the amide bonds are hydrolysed,
and a solution (hydrosylate) is obtained that contains a
mixture of all the amino acids originally present in the peptide
Analysis of the Hydrosylate of a Peptide• The amino acids in the hydrosylate are then separated by
ion-exchange chromatography in which the amino acids
separate according to their acid-base properties.
• The separation is effected by adsorbing the mixture of amino
acids on a polymeric cationic resin, and then washing the
resin with aqueous buffers of increasing pH.
• The packing (ion exchange resin) is an insoluble resin that
contains strongly acidic groups (RSO3H).
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Chromatographic separation of mixtures of Amino Acidsby Ion-exchange Chromatography
Analysis of the Hydrosylate of a Peptide
• Eluting the hydrosylate with a buffer of increasing pH causes
the individual amino acids to separate based on their
structure and basicity.
• The amino acids present in the sample are identified by
comparing the retention times on the chromatographic profile
of the hydrosylate against the 20 standard amino acids.
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Amino Acid Standard
Human Bradykinin
Aspa
rtic
acid
Thre
onin
eSe
rine
Glu
tam
ic ac
id
Gly
cine
Alan
ine
Valin
e
Met
hion
ine
Isol
euci
ne
Prol
ine
Cyst
eine
Leuc
ine
Tyro
sine
Phen
ylala
nine
Lysi
ne
Argi
nine
Hist
idin
e
pH pH 3.3 pH 4.3 pH 5.3
Serin
e
Prol
ine
Gly
cine
Phen
ylala
nine
Argi
nine
Determining the Sequence of a Peptide• Once the amino acid composition of a peptide has been
determined, then this is followed by the determination of the
sequence of the peptide: the precise order in which the
amino acid residues are connected to each other in a
peptide.
• Although, the sequencing of a long chain peptide can be a
formidable task, this has been simplified by the introduction
of a number of techniques that provide dependable
information.
• Some of these techniques such as end group analysis
(terminal residue analysis) are pivotal in determining the
terminal amino acid residues of a peptide.139:48 AM
Terminal Residue Analysis (End Group Analysis)
• Terminal residue analysis entails the identification of the
amino acid residues at the ends of a peptide chain.
• The procedures used rely upon the fact that the two ends of
the peptide are different from all the other residues and from
each other in that the N-terminal residue amigo acid contains
a free amino group, while the C-terminal residue, contains a
free carboxyl group.
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Terminal Residue Analysis
• Advantage can be taken of the functional groups on the
amino and carboxyl terminus of a peptide to mark the end
groups of the peptide.
• Since the amino group is the more nucleophilic of the two
groups, it is a more convenient and easier to mark than the
poorly nucleophilic carboxylate anion at the C-terminus.
• The amino end is marked with groups capable of surviving
the acid-catalysed hydrolysis of a peptide and ones that can
easily be detected by UV or fluorescence.
• The functionalization of the carboxyl end is almost limited to
conversion to esters or amides, but these derivatives also
cleave under acid-catalysed hydrolysis of the peptide bond.
Consequently, the labelling of the carboxyl group can not be
effectively used.159:48 AM
N-Terminal Residue Analysis: Sanger’s Protocol
• A very successful method of identifying the N-terminal
residue makes use of 2,4-dinitrofluorobenzene (DNFB)
(Sanger’s reagent), which readily undergoes aromatic
nucleophilic substitution to the free amino group of a peptide
to give a yellow N-2,4-dintrophenyl (DNP) peptide derivative
thus labelling the N-terminal amino acid with a DNP group.
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F
NO2
O2N
2,4-Dinitrofluorobenzene
Sanger's Reagent
Nucleophiles attack here through an aromatic nucleophilic addition-elimination mechanism and thereby displacing fluoride
• The reaction is carried out by mixing the peptide and 2,4-
dintrofluorobenzene (1-fluoro-2,4-dinitrobenzene) in the
presence of a weak base such as sodium carbonate.
N-Terminal Residue AnalysisSanger’s Protocol
• In the first step, the base abstracts a proton from the terminal
H3N+ group to give a free amino group which acts as a
nucleophile to displace fluoride from 1-fluoro-2,4-
dinitrobenzene.
17• The labelled peptide is then isolated.
F
NO2
O2N
2,4-Dinitrofluorobenzene
Sanger's Reagent
H3NN
NN
O
CH3CH3
H
Ph
O
H O
H
O
CH3
O
+
Val-Phe-Gly-Ala
Na2CO3
NN
NN
O
CH3CH3
H
Ph
O
H O
H
O
CH3
O
HNO2
O2N
DNP-Val-Phe-Gly-Ala (yellow solid)
N-terminus labelled peptide
N-Terminal Residue Analysis: Sanger’s Protocol
• The 2,4-dinitrophenyl-labelled peptide is then subjected to
acid hydrolysis giving the 2,4-dinitrophenyl-labelled N-
terminal amino acid and a mixture of unlabelled amino acids.
• The 2,4-DNP derivatives are generally yellow solids that
makes them easy to track in a mixture of amino acids.
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N-Terminal Residue AnalysisSanger’s Protocol
• After the hydrolysis, the 2,4-dinitrophenyl derivative of the N-
terminal amino acid is isolated and identified by comparison
of its chromatographic behaviour against that of the standard
samples of 2,4-dinitrophenyl-labelled amino acids.
• Note that only the N-terminal amino acid will bear the 2,4-
dinitrophenyl group, the other amino residues will appear in
the hydrolysis product as free amino acids.
• Sanger devised this method and used it extensively to
determine the amino acid sequence of insulin for which he
was awarded the Nobel Prize in chemistry in 1958 for this
pioneering achievement.19
N-Terminal Residue AnalysisDansylation Protocol
• A method related to the Sanger protocol employs 5-
(dimethylamino)naphthalene-1-sulphonyl chloride as the
reagent for labelling the N-terminal amino acid of the peptide:
20
• The compound is also known as dansyl chloride and the
procedure is called dansylation.
• The protocol for identification of the N-terminus amino acid
residue by dansylation is similar to Sanger’s method.
N-Terminal Residue AnalysisDansylation Protocol
• Dansyl chloride labels the peptide at the a-amino group of
the N-terminal amino acid as a sulphonamide derivative,
which is isolated and identified after hydrolysis.
21• The amount of peptide required for dansylation is very small.
N-Terminal Residue Analysis: Dansylation Protocol
• The (dimethylamino)naphthyl group imparts strong
fluorescence to dansyl derivatives, and they can be located
on paper or thin-layer chromatography plates in minute
amounts, much smaller than those required to detect 2,4-
dintrophenyl derivatives.
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NN
NN
O
CH3CH3
H
Ph
O
H O
H
O
CH3
O
Dansyl-Val-Phe-Gly-Ala
N-terminus labelled peptide
N
CH3
CH3
O
S
O
H
6 M aqueous HCl
H3N H3N H3N
Ph
O
OOH
CH3
OOH
OH
O
CH3CH3
OH + + +
Dansyl-Val Phe Gly Ala
Heat
N
N
CH3
CH3
O
S
O
H
N-Terminal Residue Analysis:Edman Degradation
• The most efficient and widely used method of N-terminal
residue analysis of peptides is the Edman degradation. It
degrades the peptide one amino acid residue at a time.
• The process is based upon the reaction between an amino
group and phenyl isothiocyanate (Edman’s reagent) to form
a substituted phenylthiourea (phenylthiocarbamoyl) (PTC
derivative).
• Mild cleavage with hydrogen chloride in an anhydrous
solvent selectively removes the N-terminal residue as a
heterocyclic derivative (phenylthiohydantoin), which is
identified, and also provides a shortened peptide chain.23
Edman Degradation of a Peptide
• In the first step, the N-terminal amino acid acts as a
nucleophile towards the C=N bond of phenyl isothiocyanate
(Edman’s reagent) to form a phenylthiourea
(phenylthiocarbamoyl) (PTC) derivative.
24
Ph N C S H2N C
H
R
HNC
O
Peptide+HN C
H
R
HNC
O
PeptideHN C
S
Ph
Phenyl isothiocyanate
Edman's reagent
Peptide Phenylthiourea derivative or(Phenylthiocarbamoyl (PTC)derivative
Mechanism:
H2N C
H
R
HNC
O
Peptide
Ph N C S+ --
N C
H
R
HNC
O
Peptide
CN
S
Ph
H
HHN C
H
R
HNC
O
Peptide
CHN
S
Ph
Nitrogen is more electronegative than sulphur
Edman Degradation of a Peptide• In the second step of the reaction, the PTC derivative is
cleaved with an anhydrous acid, commonly hydrogen
chloride in an anhydrous solvent (e.g. nitromethane) to
provide a heterocyclic derivative (phenylthiohydantoin (PTH)
derivative) and a shortened chain.
• This only cleaves the amide bond between the N-terminal
amino acid and the remainder of the peptide. No other
peptide bonds are cleaved in this step
25
H3N PeptideHN C
H
R
HNC
O
PeptideHN C
S
Ph
Shortened peptide
Phenylthiourea derivative or(Phenylthiocarbamoyl (PTC)derivative
HCl
HN CH
N O
R
S
Ph
+
Phenylthiohydantoin(PTH)
Overall Reaction
Mechanism of the Edman Degradation of a Peptide
• Mechanistically, the thiocarbonyl sulphur of the PTC
derivative attacks the carbonyl carbon of the N-terminal
amino acid.
• The N-terminal amino acid is cleaved as a thiazolone
derivative from the remainder of the peptide.
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Mechanism:
N CH
OSHN
RH
NH PeptidePh HCl
N CH
S O
R
NPh+ H2N Peptide
PTC derivative
N CH
OS
N
RH
N PeptidePh
H
H
Hindered carboxyl
Nucleophile
H
H
H
H
N CH
S O
R
NPh+ H3N Peptide
Shortened peptideThiazolone
H
H
+ Cl
H
Mechanism of the Edman Degradation of a Peptide
• The thiazolone derivative is unstable and isomerizes to a
more stable phenythiohydantoin (PTH) derivative,
27
• The specific PTH derivative formed is identified
chromatographically by comparison with PTH derivatives of
standard amino acids. This gives the identity of the original N-
terminal amino acid.
• The rest of the peptide is cleaved intact and further Edman
degradations are used successively to identify the new N-
terminus amino acid in the chain of the shortened peptide.
C-Terminal Residue Analysis Carboxypeptidase A
• While there is no efficient purely chemical technique of
sequencing amino acid residues of a peptide starting from
the C-terminus, the most widely used method is one based
on enzymatic hydrolysis.
• One group of pancreatic enzymes, the carboxypeptidases,
catalyze only the hydrolysis of the peptide bond involving the
C-terminal amino acid.
• Once the C-terminal amino acid residue has been cleaved,
yielding a peptide shorter by one amino acid, the
carboxypeptidase then catalyzes the cleavage of the peptide
bond to the new C-terminal amino acid residue.
• Eventually, the entire peptide is hydrolysed to its individual
amino acids.28
C-Terminal Residue Analysis Carboxypeptidase A
• This property can be taken advantage of in sequence
analysis through incubation of peptide with a
carboxypeptidase and subsequent monitoring of the
concentration of free amino acids as a function of time.
• Ideally, the amino acid whose concentration increases first
should be the C-terminus, and the next amino acid to appear
should be the second residue from the end.
29
H2NN
NN
O
CH3CH3
H
Ph
O
H O
H
OH
CH3
O
Val-Phe-Gly-Ala
Carboxypeptidase
H2O
H2NN
N H2N
O
CH3CH3
H
Ph
O
H OOH
CH3
OOH
Val-Phe-Gly
Alanine
+
H2NN
N
O
CH3CH3
H
Ph
O
H O
OH
Val-Phe-Gly
Carboxypeptidase
H2O
H2NN H2N
O
CH3CH3
H
Ph
O
O
OH+OH
Val-Phe Glycine
Selective Hydrolysis of Peptides• Before a large peptide can be sequenced, it is broken down
into smaller chains not longer than about 30 amino acids.
• These smaller fragments are separated, then sequenced by
the Edman degradation. The entire structure of the peptide is
deduced by fitting the short chains together in a logical
fashion.
• The partial cleavage of peptides into smaller fragments is
commonly accomplished using specific enzymes that target
bonds associated with specific amino acids.
• The isolation of these smaller fragments, coupled with the
determination of their sequences by the Edman degration
provides fragments, which when overlapped lead to
determination of the sequence of the parent peptide.
• Dietary proteins are usually not absorbed whole. They must
be digested into the component amino acids first.30
Selective Hydrolysis of Peptides
• Enzymes are selective, giving cleavage at predictable points
in the peptide chain. The most common enzymes for partial
hydrolysis of peptides are the digestive enzymes trypsin,
chymotrypsin and pepsin.
• Trypsin catalyzes only the hydrolysis of peptide bonds
involving the carboxyl group of the basic amino acid residues
lysine and arginine.
• Chymotrypsin is selective for hydrolysis of peptide bonds of
the carboxyl group of amino acids with aromatic groups in
their side chains: phenylalanine, tyrosine and tryptophan.
31
Enzyme Amino acid residue cleaved at its carboxyl side
Trypsin Lysine and Arginine
Chymotrypsin Phenylalanine, Tyrosine and Tryptophan
Pepsin Phenylalanine, Tyrosine, Tryptophan and Methionine
Determining the Primary Structure of a PeptideSelective Hydrolysis of Peptides with Trypsin
Cleavage of short peptides with trypsin
32
Determining the Primary Structure of a PeptideSelective Hydrolysis of Peptides with Chymotrypsin
Cleavage of short peptides with chymotrypsin
33
Determining the Primary Structure of a PeptideEnzymatic Hydrolysis of Bradykinin with Trypsin and Chymotrypsin
34
Determining the Primary Structure of a PeptideEnzymatic Hydrolysis of Bradykinin with Trypsin and Chymotrypsin
35
Determining the Primary Structure of a PeptideSolved Example 1
36
When an unknown peptide is hydrolysed in 6 M HCl at 110oC for 24 hours andthe amino acid composition of its hydrosylate determined, it reveals acomposition of:
Arg2, Ile2, Glu, Gly2, Leu, Lys, Phe, Pro, Ser, Trp(Note that the commas between the amino acids indicate that the sequence isunknown or unspecified and the subscripted numbers indicate the number oftimes the particular residue appears in the peptide)When the unknown peptide is dansylated and then hydrolysed, a dansylatedleucine derivative was isolated along with other undansylated amino acids.Treatment of the unknown peptide with trypsin gave the following peptidefragments, whose individual sequences were determined by Edmandegradation:
Gly-Arg; Ile-Trp-Phe-Pro-Gly-Arg; Leu-Lys; Ser-Glu-IleCleavage of the unknown peptide with chymotrypsin gave peptides whosepartial sequences are shown below:
Leu-Lys-Gly…..; Phe-Pro-Gly-Arg-Ser…(a) Using these results, determine the sequence of the unknown peptide.(b) Explain why the additional cleavage data from chymotrypsin was necessary
to define the sequence.
Determining the Primary Structure of a PeptideSolved Example 1
37
Analysis of the results of the peptide degradation and enzymatic hydrolysis
1. From the dansylation
2. From the partial hydrolysis with trypsin
It is obvious that leucine is at the N-terminus of the peptide
Since trypsin cleaves a peptide at the amide bond on the carboxyl side of the basic aminoacids (arginine and lysine), all the partial fragments directly arising from cleavage by trypsinare supposed to contain arginine or lysine at the carboxyl end, except that fragment that contains the amino acid residue at the C-terminus of the parent peptide.
So check for the partial fragment that does not contain either arginine or lysine at its carboxyl end; that fragment would be the fragment at the C-terminus of the parent peptide.
Only Ser-Glu-Ile does not contain Arg or Lys at its carboxyl end so it provides the amino acidsequence of the C-terminus of the parent peptide
(a)
(b)
Fragments obtained are: Gly-Arg; Ile-Trp-Phe-Pro-Gly-Arg; Leu-Lys; Ser-Glu-Ile
Since Leu appears only once in the composition of the peptide and a fragment Leu-Lys is obtained in the trypsin partial hydrolysis, it is safe to say that Lys in the second amino acid from the N-terminus
At this point we know that, of the four fragments, Leu-Lys is at the N-terminus and Ser-Glu-Ile is at the C-terminus. However, we can not tell the order in which the other fragements: Gly-Arg and Ile-Trp-Phe-Pro-Gly-Arg follow each other i.e. whether Gly or Ile is the third amino acid residuefrom the N-terminal.
1 2 3 4 5 6 7 8 9 10 11 12 13
Leu Lys IleGluSer
Determining the Primary Structure of a PeptideSolved Example 1
38
3. From the partial hydrolysis with chymotrypsin
This is what is resolved by the additional cleavage by chymotrypsin
Using the partial sequences from the chymotrypsin cleavage, look for points of overlap with thefragments obtained from the trypsin cleavage above.
1 2 3 4 5 6 7 8 9 10 11 12 13
Leu Lys IleGluSerFrom trypsin cleavage
From chymotrypsin cleavage Leu - Lys - Gly
Phe -Pro-Gly-Arg - Ser
From the points of overlap between the trypsin and chymotrypsin based on the knowledge that Ser and Leu appear only once in the peptide, it becomes clear that Gly is the third amino acid from the N-terminus and the rest of the chain immediately follows from the trypsin cleavage results
1 2 3 4 5 6 7 8 9 10 11 12 13
Leu Lys
IleGluSerResolved rom trypsin cleavage
From chymotrypsin cleavage
LysLeu
Gly
Phe Pro Gly Arg Ser
Unresolved from trypsin cleavageGly Arg
Ile Trp Phe Pro Gly Arg Ser
Overall sequence Leu Lys Gly Arg Ile Trp Phe Pro Gly Arg Ser Glu Ile-- - - - - - - - - -
Determining the Primary Structure of a PeptideProblem 2
39
When an unknown dipeptide was hydrolysed in 6 M HCl at 110
oC for 24 h and the hydrosylate mixture analyzed by cationic
exchange chromatography, it was found to contain Ser and Ala.
An attempted Edman degradation on the dipeptide failed. When
the dipeptide was treated with a carboxypeptidase, no free
amino acids were detected in the reaction mixture. Using these
observations, suggest a structure for the unknown dipeptide.
Determining the Primary Structure of a PeptideSolved Example 2
40
• Since the Edman reagent (Ph-N=C=S) reacts with the free
amino group of a peptide, failure of the Edman degradation
implies the absence of a free amino group in the dipeptide.
• Since carboxypeptidases cleave a peptide starting from the
amide bond of the amino acid residue with a free carboxylic
acid group, its failure suggests the absence of a free
carboxylic acid at the C-terminus.
• It appears that the dipeptide has neither a free amino group
nor a free carboxylic acid group. The simplest way of
reconciling the absences of these two groups in the free form
is if both groups were bonded to each other as a cyclic
dipeptide