Assignment of

BNB-502 3(2-1)

Topic

Submitted to:

Ms. Nida Khalid

Submitted by:

Group V

Class:

BS Biochemistry (M) Semester V

Session: 2014-2018

Department:

Applied Chemistry & Biochemistry

Hafiza Zainab 13901

Uzma Yaseen 13906

Ayesha Suleman 13907

Tayyaba Sultan 13909

Umema Mughal 13911

Faiza Tariq 13916

Muhammad Kashif 13921

Muhammad Abdullah 13922

Naveed Akram 13926

Kinza Tanveer 13952

Sheeza Tariq 13965

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 CONTENTS

➢ Introduction

➢ Amino acid Sequence

➢ Protein Sequencing

➢ Edman Reagent

➢ Protein Sequencing by Edman Degradation

▪ Protein Purification

▪ Protein Denaturation

▪ Breaking Disulfide Bonds

▪ Separation of Polypeptides

▪ Detection of Amino Terminal Residue

▪ Cleaving the Polypeptide

▪ Sequencing by Edman Reaction

▪ Ordering of Peptides

▪ Locating Disulfide Bonds

➢ Protein Sequenator

➢ Advantages

➢ Limitations

➢ References

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 INTRODUCTION

Edman degradation is a chemical method used to determine the amino acid sequence in a
peptide.

The Edman degradation chemistry was developed more than 60 years ago by Pehr Edman.
In 1950, Edman published his first paper: “Method for determination of the amino acid
sequence in peptides”.

The sequence of amino acids in a protein or peptide can be analyzed from the N-terminal by
Edman sequencing. The amino-terminal residue is labeled and cleaved from the peptide
without disrupting the peptide bonds between other amino acid residues. The labeled
amino acid is detected by various methods. This process is repeated again and again to
determine the N-terminal amino acid sequence of peptide.

The Edman degradation reaction was automated in 1967 by Edman and Beggs to speed up
the process and 100 automated devices were in use worldwide by 1973. Automated Edman
sequencers are now in widespread use, and are able to sequence peptides up to
approximately 50 amino acids long.

AMINO ACID SEQUENCE

Amino acids link to one another by peptide bonds which form through a dehydration
reaction that joins the carboxyl group of one amino acid to the amine group of the next in a
head-to-tail manner to form a polypeptide chain.

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The chain has two ends - an amine group, the N-terminus, and an unbound carboxyl group,
the C-terminus.

The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or

amine-terminus) is the start of a protein or polypeptide terminated by an amino acid with a
free amine group (-NH2).

The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, C-

terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide),
terminated by a free carboxyl group (-COOH).

When the protein is translated from messenger RNA, it is created from N-terminus to C-
terminus. By convention, peptide sequences are written (left to right) N-terminus to C-
terminus.

Each protein has a distinctive number and sequence of amino acid residues which
determines how a protein folds up into a unique three-dimensional structure and this in
turn determines the function of the protein.

PROTEIN SEQUENCING

Protein sequencing is the practical process of determining the amino acid sequence of all or
part of a protein or peptide. This may serve to identify the protein or characterize its post-
translational modifications.

Protein sequencing involves following procedures:

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 • Determining amino acid composition
• Terminal Amino Acid analysis
• Determination of Amino acid sequence
• Predicting sequence from DNA/RNA sequences

Typically, partial sequencing of a protein provides sufficient information to identify it with

reference to databases of protein sequences derived from the conceptual translation of
genes.

The two major direct methods of protein sequencing are:

Mass spectrometry
Edman degradation

Mass spectrometry methods are now the most widely used for protein sequencing and
identification but Edman degradation remains a valuable tool for characterizing a protein's
N-terminus.

EDMAN REAGENT

Phenylisothiocyanate (PITC), also known as Edman's Reagent, enables the sequential

degradation of amino acids in a polypeptide chain,
yielding primary structural information.

PITC reacts readily with amino acids at alkaline pH and

produce derivatives (PTC-amino acids) that can be
separated and quantified using reverse-phase HPLC. This
method produces stable products with all amino acids, including proline. PITC is volatile,
making it possible to remove excess reagent. Detection of picomole quantities of the
derivatives can be achieved using a UV detector at 254nm.

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 PROTEIN SEQUENCING BY EDMAN DEGRADATION

Protein sequencing by Edman degradation involves following steps:

1-Protein Purification:

Protein purification is a series of processes intended to isolate one or a few proteins from a
complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for
the characterization of the function, structure and interactions of the protein of interest.
The purification process may separate the protein and non-protein parts of the mixture, and
finally separate the desired protein from all other proteins. Following methods are used for
protein purification:

▪ Size exclusion chromatography

▪ Hydrophobic interaction chromatography
▪ Ion exchange chromatography
▪ Immunoaffinity chromatography
▪ High performance liquid chromatography
▪ SDS-PAGE
▪ 2D-PAGE

2-Protein Denaturation:

Denaturation of proteins involves the disruption and possible destruction of both the
secondary and tertiary structures. Since denaturation reactions are not strong enough to
break the peptide bonds, the primary structure (sequence of amino acids) remains the same
after a denaturation process. A protein can be denatured by

▪ Acids e.g. acetic acid

▪ Bases e.g. Sodium bicarbonate
▪ Solvents e.g. ethanol & alcohol
▪ Chaotropic agents (weaken hydrophobic effect) e.g. Urea 6 – 8 mol/l
▪ Temperature

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3-Breaking Disulfide Bonds:

Disulfide bonds interfere with the sequencing procedure. A cystine residue that has one of
its peptide bonds cleaved by the Edman procedure may remain attached to another
polypeptide strand via its disulfide bond. Disulfide bonds also interfere with the enzymatic
or chemical cleavage of the polypeptide. Two approaches to irreversible breakage of
disulfide bonds are outlined below:

4-Separation of Polypeptides:

Some proteins contain two or more separate polypeptide chains, or subunits, which may be
identical or different. In this case polypeptides are separated by purification methods.

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5-Detection of Amino-Terminal Residue:

Several protocols are available to label and identify the amino-terminal amino acid residue.
Sanger developed the reagent 1-fluoro-2,4-dinitrobenzene (FDNB) for this purpose; other
reagents used to label the amino-terminal residue, dansyl chloride and dabsyl chloride, yield
derivatives that are more easily detectable than the dinitrophenyl derivatives. After the
amino-terminal residue is labeled with one of these reagents, the polypeptide is hydrolyzed
to its constituent amino acids and the labeled amino acid is identified.

6-Cleaving the Polypeptide Chain:

The overall accuracy of amino acid sequencing generally declines as the length of the
polypeptide increases. The very large polypeptides found in proteins must be broken down
into smaller fragments (approximately 50 amino acid each) to be sequenced efficiently.
Several methods can be used for fragmenting the polypeptide chain. Enzymes called
proteases cleave only the peptide bond adjacent to particular amino acid residues and thus
fragment a polypeptide chain in a predictable and reproducible way e.g. trypsin cleaves at
arginine or lysine while cyanogen bromide cleaves where methionine residue is present. A
number of chemical reagents such as cyanogen bromide can also be used for this purpose.
These fragments are separated by chromatographic and electrophoretic methods.

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7-Sequencing by Edman Reaction:

The peptides are first immobilized by absorption on a chemically modified glass or by

electroblotting onto a porous polyvinylidene fluoride (PVDF) membrane. The peptide is
reacted with phenylisothiocyanate under mildly alkaline conditions, which converts the
aminoterminal amino acid to a phenylthiocarbamoyl (PTC) adduct. The peptide bond next to
the PTC adduct is then cleaved in a step carried out in anhydrous trifluoroacetic acid, with
removal of the amino-terminal amino acid as an anilinothiazolinone derivative. The
derivatized amino acid is extracted with organic solvents, converted to the more stable
phenylthiohydantoin derivative by treatment with aqueous acid, and then identified. The
use of sequential reactions carried out under first basic and then acidic conditions provides
control over the entire process. Each reaction with the aminoterminal amino acid can go
essentially to completion without affecting any of the other peptide bonds in the peptide.
After removal and identification of the aminoterminal residue, the new amino-terminal
residue so exposed can be labeled, removed, and identified through the same series of
reactions. This procedure is repeated until the entire sequence is determined.

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8-Ordering of Peptides:

The amino acid sequences of each fragment obtained by the two different cleavage
procedures are examined. Pre-identified amino-terminal residue provides information about
fragment derived from the amino terminus. Overlapping peptides sequences obtained from
the second fragmentation yield the correct order of the peptide fragments produced in the
first. If the second cleavage procedure fails to establish continuity between all peptides from
the first cleavage, a third or even a fourth cleavage method must be used to obtain a set of
peptides that can provide the necessary overlap(s).

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9-Locating Disulfide Bonds:

If the primary structure includes disulfide bonds, their locations are determined in an
additional step after sequencing is completed. A sample of the protein is again cleaved with
a reagent such as trypsin, this time without first breaking the disulfide bonds. The resulting
peptides are separated by electrophoresis and compared with the original set of peptides
generated by trypsin. For each disulfide bond, two of the original peptides will be missing
and a new, larger peptide will appear. The two missing peptides represent the regions of the
intact polypeptide that are linked by the disulfide bond.

PROTEIN SEQUENATOR

The Edman degradation reaction was automated in 1967 by Edman and Beggs to speed up
the process and 100 automated devices were in use worldwide by 1973. Protein sequenator
is a machine that performs Edman degradation in an automated manner. A sample of the
protein or peptide is immobilized in the reaction vessel of the protein sequenator and the
Edman degradation is performed. Each cycle releases and derivatises one amino acid from
the protein or peptide's N-terminus and the released amino-acid derivative is then identified
by HPLC. The sequencing process is done repetitively for the whole polypeptide until the
entire measurable sequence is established or for a pre-determined number of cycles.
Modern sequenators achieve efficiencies of better than 99% per cycle, permitting the
sequencing of more than 50 contiguous amino acid residues in a polypeptide.

ADVANTAGES

✓ The procedure can be achieved with very minute amounts of peptide, usually
amounts on the order of 10-100 picomoles will allow for successful completion.
✓ 100% surety of finding protein sequence.
✓ Edman sequencing can be performed on PVDF blots from 1D and 2D gels. This
enables N-terminal sequencing of proteins in mixtures.

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 LIMITATIONS

❖ A major drawback to this technique is that the peptides being sequenced in this
manner cannot have more than 50 to 60 residues (and in practice, fewer than 30).
❖ A pure sample of peptide is necessary for accuracy
❖ As Edman degradation proceeds from the N-terminus of the protein, it will not work
if the N-terminus has been chemically modified (e.g. by acetylation)
❖ Edman degradation is generally not useful to determine the positions of disulfide
bridges.
❖ It is time consuming process. It requires approximately 40 to 60 minutes for one
amino acid detection.

REFERENCES

➢ Edman P., Högfeldt E., Sillén LG., Kinell P. “Method for determination of the amino
acid sequence in peptides”. Acta Chem. Scand. 4: 283–293, 1950.
➢ Lehninger's Principles Of Biochemistry 2008 [4th Edition]
➢ Application note: Applications of N-terminal Edman Sequencing #201303, v10-0916
➢ http://www.biotecharticles.com/Genetics-Article/Edman-Degradation-For-Protein-
Sequencing-340.html
➢ https://en.wikipedia.org/wiki/Protein_sequencing#Edman_degradation
➢ https://en.wikipedia.org/wiki/Edman_degradation
➢ https://en.wikibooks.org/wiki/Proteomics/Protein_Primary_Structure/Sequencing_
Methods
➢ https://www.thermofisher.com/order/catalog/product/26922
➢ https://www.thebalance.com/methods-for-protein-purification-375683

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