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.

18

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.

22

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