The document discusses various techniques for amino acid analysis. It defines amino acids and explains that 20 are encoded by the genetic code. There are essential and non-essential amino acids. Amino acid analysis involves hydrolyzing proteins into individual amino acids, separating them using chromatography, and quantifying amounts. Common techniques discussed include determining amino acid composition, Edman degradation, and mass spectrometry.

1. Prepared By:
PARTH
Guided By:
Rajesh Parmar
Apmc college of pharmaceutical education and
research.
1

2.  Introduction
 Amino acid analysis
 General techniques for analysis of amino acids
 Method used for amino acid analysis
 Reference.
2

3.  Definition: “Amino acids are organic compounds
containing an amine group and a carboxylic acid
group, a side chain that varies between different
amino acids.”
3

4.  One amino acid molecule can react with another and become
joined through an amide linkage.
 This polymerization of amino acids creates proteins, this
condensation reaction yields the newly formed peptide bond
and a molecule of water.
4

5.  20 amino acid are encoded by genetic code.
 Several amino acids are called "essential" for humans
because they cannot be created from other compounds by
the human body, and so must be taken in as food.
 Non essential amino acids which the body can synthesized,
Alanine, Proline, Asparagine, Aspartic acid, Cysteine,
Tyrosine, Serine, Glycine, Glutamine and Glutamic acid.
 Essential amino acids which the body cant synthesize are,
Isoleucine, leucine, Lysine, Methionine, Valine, Phenyl
alanine, Threonine, Tryptophan, Histidne and Arginine.
5

6.  Amino acid analysis refers to the methodology used to
determine the amino acid composition or content of
proteins, peptides, and other pharmaceutical
preparations.
 Proteins and peptides are macromolecules consisting of
covalently bonded amino acid residues organized as a
linear polymer.
 The sequence of the amino acids in a protein or peptide
determines the properties of the molecule.
6

7.  Amino acid analysis can be used to
 Quantify protein and peptides,
 Determine the identity of proteins or peptides based on
their amino acid composition,
 Support protein and peptide structure analysis,
 Evaluate fragmentation strategies for peptide mapping,
 Detect atypical amino acids that might be present in a
protein or peptide.
 It is necessary to hydrolyze a protein/peptide to its
individual amino acid constituents before amino acid
analysis.
7

8.  General techniques for analysis of amino acids
are:
1. Determining amino acid composition
Hydrolysis
Separation
Quantitative analysis
2. N-terminal amino acid analysis
3. C-terminal amino acid analysis
8

9. 4. Edman degradation
5. Mass spectrometry
6. Predicting protein sequence from DNA/RNA
sequences
 The two major direct methods of protein sequencing
are mass spectrometry and the Edman degradation
reaction
9

10. 1.) Determination of amino acid composition
Hydrolysis
 Hydrolysis is done by heating a sample of the protein in 6M
hydrochloric acid to 100-110 oC for 24 hr or longer.
 Proteins with many bulky hydrophobic groups may require
longer heating periods.
10

11.  However, these conditions are so vigorous that some amino
acids (serine, threonine, tyrosine, tryptophan, glutamine and
cystine) are degraded.
 Rastall suggests a variety of reagents to prevent or reduce
degradation - thiol reagents or phenol to protect tryptophan
and tyrosine from attack by chlorine, and pre- oxidising
cysteine.
11

12. H O H H O H H O H H O H H O H H O
H 3N C C N C C N C C N C C N C C N C C O
C H3 C H 2O H C H2 C H(C H 3 ) 2 H C H 2C H 2S C H 3
A S F V G M
H 3 O , h eat
(tot al hydr olysis)
H O H O H O H O H O H O
H 3N C C O + H 3N C C O + H 3N C C O + H 3N C C O + H3 N C C O + H 3N C C O
C H3 C H 2O H C H2 C H(C H 3 ) 2 H C H 2C H 2S C H 3
A S F V G M
(equ imolar mixture of
A, S , F , V , G , an d M )
Hydrolysis: conversion of a peptide into a mixture of its component amino acids
12

13.  Separation
 The amino acids can be separated by ion-exchange
chromatography or hydrophobic interaction chromatography.
 Amino acids will be eluted when the pH reaches their respective
isoelectric points. The latter technique may be employed through
the use of reversed phase chromatography.
 Many commercially available C8 and C18 silica columns have
demonstrated successful separation of amino acids in solution in
less than 40 minutes through the use of an optimised elution
gradient.
13

14.  Quantitative analysis
 Once the amino acids have been separated, their respective
quantities are determined by adding a reagent that will form a
coloured derivative.
 If the amounts of amino acids are in excess of 10 nmol,
ninhydrin can be used for this - it gives a yellow colour
 when reacted with proline, and gives a purple colour.
 The concentration of amino acid is proportional to the
absorbance of the resulting solution.
14

15.  Sanger's method of peptide end-group analysis:
A. derivatization of N-terminal end with Sanger's reagent
2-4 dinitroflorobenzen(DNFB),
B. Total acid hydrolysis of the dinitrophenyl peptide
15

16. H O H H O H H O H H O H H O H H O
H 3N C C N C C N C C N C C N C C N C C O
C H3 C H 2O H C H2 C H (C H 3 ) 2 H C H 2C H 2S C H 3
A S F V G M
S anger's R eagent
(2,4- dinitrof luor obe nzene)
H H O H H O H H O H H O H H O H H O
O 2N N C C N C C N C C N C C N C C N C C OH
C H3 C H 2O H C H2 C H (C H 3 ) 2 H C H 2C H 2S C H 3
NO2
16

17.  The number of methods available for C-terminal amino acid
analysis is much smaller than the number of available
methods of N-terminal analysis.
 The most common method is to add carboxypeptidases to a
solution of the protein, take samples at regular intervals, and
determine the terminal amino acid by analysing a plot of
amino acid concentrations against time.
17

18. Carboxypeptidase: C-terminal AA Analysis
H O H H O H H O H H O H H O H H O
H 3N C C N C C N C C N C C N C C N C C O
C H3 C H 2O H C H2 C H (C H 3 ) 2 H C H 2C H 2S C H 3
A S F V G M
C a rbox ype pt ida se
H O H H O H H O H H O H H O H O
H 3N C C N C C N C C N C C N C CO + H 3N C CO
C H3 C H 2O H C H2 C H (C H 3 ) 2 H C H 2C H 2S C H 3
M
C a rbox ype pt ida se
H O H H O H H O H H O H O
H 3N C C N C C N C C N C CO + H3 N C CO
C H3 C H 2O H C H2 C H (C H 3 ) 2 H
G
18

19.  Edman degradation, developed by Pehr Edman, is a very
important reaction for protein sequencing, because it allows
the ordered amino acid composition of a protein to be
discovered.
 Now a day, Automated Edman sequencers are used, and are
able to sequence peptides up to approximately 50 amino
acids long.
19

20.  A reaction scheme for sequencing a protein by the Edman
degradation follows - some of the steps are describe below.
1. Break any disulfide bridges in the protein with an oxidising
agent like per formic acid or reducing agent like 2-
mercaptoethanol.
2. Separate and purify the individual chains of the protein
complex, if there are more than one. Determine the amino
acid composition of each chain.
3. Determine the terminal amino acids of each chain. 20

21. 4. Break each chain into fragments under 50 amino acids
5. Separate and purify the fragments.
6. Determine the sequence of each fragment.
7. Repeat with a different pattern of cleavage.
8. Construct the sequence of the overall protein.
21

22.  Phenylisothiocyanate is reacted with an uncharged terminal
amino group, under mildly alkaline conditions, to form a
cyclical phenylthiocarbamoyl derivative. Then, under acidic
conditions, this derivative of the terminal amino acid is
cleaved as a thiazolinone derivative.
 The thiazolinone amino acid is then selectively extracted
into an organic solvent and treated with acid to form the
more stable phenylthiohydantoin (PTH)- amino acid
derivative that can be identified by using chromatography or
electrophoresis.
22

23. 23

24. 24

25.  The amino acid sequence of a protein can also be determined
indirectly from
 the mRNA or,
 the DNA that codes for the protein. (e.g. prokaryotes).
25

26.  This can then be used to isolate the mRNA coding for the
protein, which can then be replicated in a polymerase chain
reaction to yield a significant amount of DNA, which can
then be sequenced relatively easily.
26

27.  Gel electrophoresis
 Electrophoresis is a separation technique based on the
movement of charged ions under the influence of an
electrical field. This technique is primarily used for the
separation of amino acids and peptides on the basis of their
charge.
 In this experiment of separating amino acids, a phosphate
buffer (pH 6) will be used as the conducting liquid and
cellulose as the supporting medium.
27

28.  Polyacrylamide Gel Electrophoresis (PAGE)
PAGE (Polyacrylamide Gel Electrophoresis) SDS(sodium
dodecyl sulfste) is the most widely used analytical method to
resolve separate components of a protein mixture based on their
size
H O H O H O
M ixture of : H 3N C C O + H 3N C CO + H3 N C CO
bu ffered a t pH 6.0 C H 2C O 2 C H3 C H 2 C H 2 C H 2 C H2 N H 3
D ( pI= 2.8) A (pI= 6.0) K ( pI= 9.7)
28

29.  Before current is turned on:
K
A
D
 After current is turned on:
K
A
D
29

30.  The polymerase chain reaction (PCR) is a scientific technique in
molecular biology to amplify a single or a few copies of a piece of
DNA across several orders of magnitude, generating thousands to
millions of copies of a particular DNA sequence
 The polymerase chain reaction (PCR) is an in vitro technique
which allows the amplification of a specific deoxyribonucleic acid
(DNA) region that lies between two regions of known DNA
sequences..
30

31.  This helix comprises of two single strands of DNA running
antiparallel to each other and held together non-covalently by
hydrogen bonds.
 The hydrogen bonds form between the complementary bases,
i.e., adenine (A) with thymine (T) and guanine (G) with
cytosine (C).
31

32.  X-ray crystallography is a technique which is widely used to
determine structures of proteins.
 It exploits the fact that X-rays are scattered or diffracted in a
predictable manner when they pass through a protein crystal.
X-rays are diffracted when they encounter electrons, so the
nature of the scattering depends on the number of electrons
that are present in each atom and the organization of the
atoms in space.
32

33.  In principle X-ray analysis of very large and
complex organic molecules like proteins is possible
but the mathematical analysis of the diffraction
patterns is very complex because large number of
atoms in the molecule may yield thousands of
diffraction spots.
 X-ray crystallography is the primary method used to
determine protein structures
33

34.  Has allowed determination of structures as large as viruses
and ribosomes to be completed
 X-ray methods are fast and depend on computers and robots
 X-ray structures are generally more accurate than NMR
structures.
34

35.  Isoelectric focusing takes place in a pH gradient and is limited to
molecules which can be either positively or negatively charged
(amphoteric molecules), like proteins, enzymes and peptides.
 Each amino acid has an isoelectric point(pI) numerically equal to
the pH at which the zwitterion concentration is at a maximum.
O OH O O O O
C C C
H OH
H 3N H H 3N H H 2N H
R R R
@ pH < pI @ pH = pI @ pH > pI
35

36. Equipment for Isoelectric focusing (IEF):
(IEF System) Protein IEF Cell
Amersham Pharmacia Biotech Inc. Bio-Rad Laboratories
36

37.  This is a technique for detecting atoms that have nuclei that
possess a magnetic moment. These are atoms in which either
the protons or the neutrons or both are odd in number.
 1H atom (one proton), 13C and 14N are some such isotopes
of hydrogen, carbon and nitrogen respectively
 In principle, it is possible to obtain a unique signal for each
hydrogen atom except in case of those which are chemically
equivalent. However, this problem has been bypassed by
using 2D NMR spectroscopy.
37

38.  A COSY (correlated spectroscopy) experiment gives
peaks between hydrogen atoms that are covalently
connected through one or two other atoms, for
example, the hydrogen atoms connected to nitrogen
or carbon atoms within the same amino acid residue.
 A NOE (nuclear Overhauser spectroscopy)
spectrum, on the other hand gives peaks between
pairs of hydrogen atoms that are close together in
space even if they are from amino acid residues that
are quite distant in the primary sequence.
38

39.  Mass spectroscopy
 This process, known as matrix-assisted laser
desorption/ionization mass spectrometry, or MALDI
MS, has been successfully used to measure the mass of
a wide range of macromolecules.
 A solution of analytes is passed through a charged
needle that is kept at a high electrical potential,
dispersing the solution into a fine charged
microdroplets.
39

40.  This creates a spectrum of species with different
mass-to-charge ratios.
 Each successive peak corresponds to a species that
differs from that of its neighboring peak by a
charge difference of 1 and a mass difference of 1 (1
proton).
 The mass of the protein can be determined from
any two neighboring peaks. The measured m/z of
one peak is
40

41. where
 M is the mass of the protein,
 n2 is the number of charges, and
 X is the mass of the added groups (protons in this case).
41

42. LC/MS
42

43. A protein solution is dispersed into highly charged droplets by passage
through a needle under the influence of a high-voltage electric field. The
droplets evaporate, and the ions (with added protons in this case) enter the
mass spectrometer for m/z measurement
43

44. Amino acid sequence analysis by MS - an example
The spectrum generated (b) is a family of peaks, with each successive
peak (from right to left) corresponding to a charged species increased by
1 in both mass and charge. 44

45.  Chromatographic method have been used to determination
of the purity of small organic molecules and protein
 the result depend on size and /or shape , charge, and
hydrophobicity of the protein
 RP-HPLC reveres phase high performance liquid
chromatography)
 IEC (ion exchange chromatography)
 SEC (size exclusion chromatography)
 HIC (hydrophobic interaction chromatography)
45

46. Principle:
 A portion of the material to be separated will
be found in the mobile phase
 A portion will be adsorbed to the solid
adsorbent particles.
46

47. Calculate the Rf value
Distance of the amino acid from the origin
Rf =
Distance of the solvent front from the origin
D1
Rf=
D2
47

48.  Negatively charged proteins will bind to positively
charged column materials (anion exchangers), while
positively charged or neutral proteins will flow
through the column. The more negatively charged,
the tighter it will bind and the higher the salt it will
take to elute it from the column
 Some highly charged proteins, where the charge is
48
not evenly distributed, can bind to both anion and

49. 49

50. SO 3 K (stron gly r eta ine d)
sulfon ate d A (slightly re ta ine d, &
SO 3
pol ystyre ne
D (unre ta ine d)
SO 3
D A K
50

51. REVERSE PHASE & HYDROPHOBIC
INTERACTION
 separation of hydrophobic proteins
 stationary phase is non polar liquid (hydrophobic)
 polar mobile phase
 polarity of mobile phase reduced to proteins.
 hydrophobic interaction chromatography phenyl
sephadex and octal sephadex is used with weak
interaction to prevent denaturation
51

52. The PTC-amino acids are separated on a reverse
phase C18 silica column and the PTC chromophore
is detected at 254 nm. All of the amino acids will
elute in approximately 25 minutes.
Many commercially available C8 and C18 silica
columns have demonstrated successful separation of
amino acids in solution in less than 40 minutes through
the use of an optimised elution gradient.
52

53. 53

54. 1. Lehninger Principles of Biochemistry, 4th Edition Chapter 9.
2. Henry Jakubowski. Biochemistry Online, chapter 2 B.[1]
3. Hanno Steen & Matthias Mann. The abc's (and xyz's) of peptide
sequencing. Nature Reviews Molecular Cell Biology, 5:699-711,
2004.
4. Sergio Marceline Michael W. King. Analysis of protein.[2]
5. R A Rastall. Investigating protein structure and function.[3]
6. Alberts Bray Johnson Lewis Raff Roberts & Walter. 1998.
Essential Cell Biology: An Introduction to the Molecular Biology
of the Cell. Garland Publishing, New York.
7. http://www.pafko.com/history/h_intro.html

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