this is a ppt on the topic..Primary structure of proteins.

1. PROTEINS: PRIMARY
STRUCTURE
PRESENTATION BY IFRAH NASEE
MSC BIOCHEMISTRY (SEM-1)

2. CONTENTS
INTRODUCTION
PRIMARY STRUCTURE OF
PROTEINS
DETERMINATION OF
PRIMARY STRUCTURE
DETERMINATION OF
AMINO ACID
COMPOSITION
DETERMINATION OF
AMINO ACID SEQUENCES
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3. Introduction
Proteins are the most abundant organic molecules of the living system. They occur in every part of
the cell and constitute about 50% of the cellular dry weight. Proteins form the fundamental basis of
structure and function of life.
The term protein is derived from a Creek word proteiog meaning holding the first place.
Proteins perform a great variety of specialized and essential functions in the living cells
Elemental composition of proteins
Proteins are predominantly constituted by five major elements in the following proportion:
Carbon: 50 - 55%
Hydrogen: 6 - 7.3%
Oxygen : 19 - 24%
Nitrogen: 13 - 19%
Sulfur : 0 – 4%
Besides the above, proteins may also contain other elements such as P, Fe, Cu, l, Mg, Mn, Zn etc
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4. Structure of proteins
Proteins are polymers of amino acids
Proteins on complete hydrolysis (with concentrated HCI for several hours) yield L-alpha-amino
acids. This is a common property of all the proteins. Therefore, proteins are the polymers of L-
alpha-amino acids.
The structure of proteins is rather complex which can be divided into 4 levels of organization:
1. Primary structure : The linear sequence of amino acids forming the backbone of proteins
(polypeptides).
2. Secondary structure: The spatial arrangement of protein by twisting of the polypeptide chain.
3. Tertiary structure: The three dimensional structure of a functional protein.
4. Quaternary structure : Some of the proteins are composed of two or more polypeptide chains
referred to as subunits. The spatial arrangement of these subunits is known as quaternary
structure.
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5. Diagrammatic representation of
protein structure:
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6. PRIMARY STRUCTURE OF
PROTEINS
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7. Each protein has a unique sequence of amino acids which is
determined by the genes contained in DNA. The primary structure of
a protein is largely responsible for its function. A vast majority of
genetic diseases are due to abnormalities in the amino acid
sequences of proteins i.e. changes associated with primary structure
of protein. The amino acid composition of a protein determines its
physical and chemical properties.
The amino acids are held together in a protein by covalent peptide
bonds or linkages.
When the amino group of an amino acid combines with the carboxyl
group of another amino acid, a peptide bond is formed
A dipeptide will have two amino acids and one peptide (not two)
bond. Peptides containing more than 10 amino acids (decapeptide)
are referred to as polypeptides.
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8. The peptide bond is rigid and planar with partial double bond in character.
lt generally exists in trans configuration. Both -C=O and -NH groups of
peptide bonds are polar and are involved in hydrogen bond formation.
Dimensions of a Peptide chain : The two adjacent alpha-carbon atoms
are placed at a distance of 0.36 nm. The interatomic distances and bond
angles are also shown in this figure
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9. Determination of primary
structure of protein
The primary structure comprises the
identification of constituent amino
acids with regard to their quality,
quantity and sequence in a protein
structure. A pure sample of a protein
or a polypeptide is essential for the
determination of primary structure
which involves 3 stages
1. Determination of amino acid
composition.
2. Degradation of protein or
polypeptide into smaller fragments.
3. Determination of the amino acid
sequence.
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10. Why do we need to determine the
primary structure of protein?
Since proteins play an important role in enzymes,
hormones, immune system, in expression of genetic
information transporters etc and to synthesize the
above products by using the application of
biotechnology we need to determine the primary
structure of protein.
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11. 1.Determination of amino acid composition
in a protein
The protein or polypeptide is completely hydrolyzed to liberate the amino acids which are
quantitatively estimated. The hydrolysis may be carried out either by acid or alkali treatment or by
enzyme hydrolysis. Treatment with enzymes, however results in smaller peptides rather than amino
acids.
• The first step in determining the primary structure of a polypeptide is to identify and quantitate its
constituent amino acids.
• Cleavage of disulphide bonds by the 2- Ercaptoethanol
• A purified sample of the polypeptide to be the analyzed is first hydrolyzed by the strong acid (6M
HCL) at 110 degree Celsius for 24 hours.
• This treatment cleaves the peptide bonds and releases the individual amino acids ,which can be
separated by cation exchange chromatography.
• The separated amino acids contained in the elute from the column are quantitated by heating them
with Ninhydrin a reagent that forms a purple compound with most amino acids, ammonia and
amines.
• The amount of each amino acid is determined by spectrophotometry by measuring the amount of
light absorbed by the ninhydrin derivative.
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12. Several methods are used :
Acid hydrolysis – Purified sample to be analyzed is first hydrolyzed by 6N HCL and
heated at 110-120 degree Celsius for 24 hours in a sealed vessel. The peptide
bonds are broken and the hydrolysis is analyzed by HPLC to determine the
composition.
It degrades serine, threonine, tyrosine, tryptophan.
Alkaline hydrolysis – Purified sample to be analyzed is hydrolyzed by a strong
base like NaOH and hydrolysate is examined. It does not destroy tyrosine,
tryptophan, and glutamine but it destroys serine, threonine, and arginine.
Enzymatic hydrolysis – To cleave the proteins in a small number of peptide
fragments. Cyanogen bromide (CNBr) splits polypeptide chain only on the carboxylic
side of methionine residues.
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13. 2.Determination of amino acid sequence
End group analysis- Identification of N terminal and C terminal amino acids in
a polypeptide chain is called End group analysis.
Identification of N terminal:
• SANGER’S METHOD
• EDMAN’S DEGRADATION METHOD
• DANSYL CHLORIDE METHOD
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14. Sanger’s method
• This was the first technique to determine the
sequence of proteins.
• Sanger’s reagent- 2,4 Dinitrofluorobenzene
• Sanger’s reagent derivatives are the amino terminal
residues
• The first protein to be sequenced by the method is
insulin by FREDRICK SANGER for which he got the
nobel prize in 1958.
• Only dipeptides or tripeptides can be sequenced.
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15. • 2,4 Dinitrofluorobenzene is reacted with amino group of a peptide or a protein to form 2,4 dinitrophenyl
derivative of N- terminal amino acid which is yellow in colour.
• The treated peptide is then subjected to acid hydrolysis which cleaves all the peptide bonds except the bond
between 2,4 DNF and NH2 GROUP which is resistant to acid hydrolysis.
• Separated by chromatography
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16. Edman’s degradation method
• The process was developed by Pehr Edmen
• Edman’s reagent- phenyl isothiocyanate
• It is a technique for identifying specific amino acid at each position in the
peptide chain beginning at the amino terminal end.
• Edman’s technique can sequence many residues (5-40) of a single
polypeptide sample
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17. Method
Phenyl isothiocyanate (PITC)
react with amino group of a
polypeptide under mild alkaline
conditions to form a
corresponding phenylthiol-
carbamoyl peptide
It involves a controlled stepwise
cleavage of the peptide
ADVANTAGE: This method over
sanger’s method is that the
remaining peptide after the
removal of the N terminal amino
acid is not hydrolyzed and can
be used again to detect the next
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18. Dansyl chloride method
Dansyl chloride- 5’dimethyl-1-
naphthalene-sulphonoyl-
chloride
NH2 terminal is reacted with
dansyl chloride which is a
fluorescent compound to form a
fluorescent dansyl amino acid
derivative
This derivative is removed from
polypeptide by hydrolysis.
Amino terminal residue is
separated by chromatography
ADVANTAGE- It is a sensitive
method and can detect
picomole quantity
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19. Identification of C- terminal amino acids
 Akabori method
 By carboxypeptidase
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20. Akabori method
• This method involves
treatment with hydrazine
• In this, identification of C
terminus amino acids involves
the heating of a linear peptide
in the presence of anhydrous
hydrazine in a sealed tube for
several hours.
• The amino group of each
peptide bond reacts with
hydrazine to form the
corresponding amino acid
hydrazide
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21. The application of microwave irradiation to the Akabori reaction
was investigated. It was found that microwave irradiation
reduced the time required for the completion of the Akabori
reaction from hours to minutes.
This approach provided information not only about the C
terminus but also the N terminus
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22. By Carboxypeptidases
Carboxypeptidases cleave a polypeptide from C terminal removing one amino acid at a
time by cleaving the peptide bond
Removal of one amino acid leaves behind a new polypeptide which is the target of
carboxypeptidase
Carboxypeptidase that have a stronger preference for those amino acids containing
aromatic or branched hydrocarbon chains are called carboxypeptidase A
Carboxypeptidases that cleave positively charged amino acids (arginine, lysine) are
called Carboxypeptidases
Plants contain carboxypeptidases C that liberates the amino acid proline as well as being
able to release many of the other proteins and amino acids.
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23. Cleavage of the polypeptide into smaller
fragments :Enzymatic method
• To cleave the proteins into a small number of pure
fragments.
• Example: Cyanogen Bromide (CNBr) splits
polypeptide chain only on the carboxylic side of the
methionine residues
• Highly specific cleavage is also obtained by trypsin, a
proteolytic enzyme secreted by pancreas
• Trypsin cleaves polypeptide chain on the carboxylic
side of arginine residues
• Peptides obtained specific chemical or enzyme
cleavage are separated by some type of
chromatography.
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26. Thank you
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