1. Paul D. Adams • University of Arkansas
Mary K. Campbell
Shawn O. Farrell
http://academic.cengage.com/chemistry/campbell
AMINO ACIDS AND PEPTIDES
Jesson B. Belen – Negros Oriental State University
4. - Organic compounds of high molecular weight
made up of α-amino acids joined by peptide
linkage
General Characteristics
1. Most important among all biological
products being the fundamental constituents
of the protoplasm of the cell
2. Most complex and most diverse in chemical
composition, conferring upon the different
tissues a sort of biological specificity
3. Supplies the body not only for materials, for
heat and energy but also for growth and
repair
PROTEINS
5. AMINO ACIDS SHARE MANY FEATURES,
DIFFERING ONLY AT THE R SUBSTITUENT
5
6. 1. All amino acids, except glycine are optically
active due to the presence of an asymmetric
carbon atom in the molecule
Stereochemistry- 3-D shape of the molecule
- objects possess mirror images
Mirror images can be superimposable or
nonsuperimposable to each other
Ex. Mugs, Hands,
PROPERTIES OF AMINO ACIDS
7. - Nonsuperimposable mirror images are said to
be “CHIRAL”
- A molecule with a chiral center is said to be
optically active and can have sterioisomers
- A chiral center is a carbon bonded to four
different groups
PROPERTIES OF AMINO ACIDS
8. - The number of possible sterioisomers of a
given chiral molecule is equal to “n2”, where
“n” is the number of stereogenic center/chiral
center
- For a given amino acid with 1 chiral center,
there are two possible sterioisomers; L- and D-
isomers
PROPERTIES OF AMINO ACIDS
11. Common amino acids can be placed in
five basic groups depending on their R
substituents:
• Nonpolar, aliphatic (7)
• Aromatic (3)
• Polar, uncharged (5)
• Positively charged (3)
• Negatively charged (2)
CLASSIFICATIONS OF AMINO ACIDS
22. 1. In the following group, identify the
amino acids with nonpolar side chains
and those with basic side chains:
alanine, serine, arginine, lysine,
leucine, and phenyalanine.
2. The pKa of the side-chain imidazole
group of histidine is 6.0. What is the
ratio of uncharged to charged side
chains at pH 7.0?
APPLY YOUR KNOWLEDGE
23. 3. Amino acids can act as either an acid
or a base.
- α-Amino group and α-carboxyl group
- At low pH, these groups are fully
protonated
- As the pH increases the carboxyl group
loses fist then the amino group follows.
PROPERTIES OF AMINO ACIDS
25. TITRATION CURVE OF AMINO ACID
Isoelectric Point (pI)- the pH at which the
amino acid has no net charge
pI = (pKa1 + pKa2)/2
Where pKa1 and pKa2 are the two pH
ranges at which the zwitterion occurs
28. APPLY YOUR KNOWLEDGE
1. Which of the following amino acids has
a net charge of +2 at low pH? Which
has a net charge of -2 at high pH? D, A,
R, E, L, K.
2. What is the pI of glutamic acid?
3. What is the pI of lysine?
30. APPLICATION
Problem: Paper electrophoresis at pH 6.0
was carried out on a mixture of G, A, E, K, R,
S.
a. Which compound (s) moved fastest
toward the anode?
b. Which moved fastest toward the cathode?
c. Which remained at or near the origin?
31. PROPERTIES OF AMINO ACID
4. Amino acids react to form peptide bonds.
The peptide group is planar as a result of
resonance stabilization.
- Water is eliminated in the process
32. PROPERTIES OF AMINO ACID
- Amino acids can form dipeptide,
tripeptide, and so on.
33. PROPERTIES OF AMINO ACID
1. Draw a polypeptide that contains amino
acids, D, A, R, E, L, and K and name it.
2. Determine the isoelectric point (pI) of the
polypeptide
34. PROPERTIES OF AMINO ACID
5. Amino acids can form esters with
alcohols and can be acetylated, benzylated
or methylated
Draw the structures of Aspartame (L-
aspartyl-L-phenylalanine, and D-aspartyl-D-
phenylalanine)
36. SMALL PEPTIDES WITH
PHYSIOLOGICAL ACITVITY
2. Glutathione- scavenger for oxidizing
agents
- γ-glutamyl-L-cysteinylglycine
Draw the structure of glutathione and
identify the disulfide bond or linkage
37. SMALL PEPTIDES WITH
PHYSIOLOGICAL ACITVITY
3. Enkephalins –pentapeptides
- analgesics (pain relievers)
-Tyr-Gly-Gly-Phe-Leu
-Tyr-Gly-Gly-Phe-Met
38. QUIZ
1. Draw the structure of the two
sterioisomers of an amino acid Serine.
2. Predict the structure of the following
amino acids at pH=7: E, L, T, R
3. What is the pI of Arginine
4. Draw a fully protonated tetrapeptide that
contains, E, L, T, R amino acids and name
the tetrapeptide
5. Determine the pI of the tetrapeptide in
number 4.
39. SUPERSECONDARY STRUCTURE AND
DOMAINS
βαβ unit – two parallel strands of β-sheets
connected by a stretched of α-helix
αα unit (helix-turn-helix) – consists of two
anti-parallel α-helices
β-meander – antiparallel sheet is formed by
a series of tight reverse turns connecting
stretches of the polypeptide chain
43. COLLAGEN TRIPLE HELIX
Collagen –
component of bone
and connective
tissues. Most
abundant protein in
vertebrates. Has
water-insoluble
fibers of great
strength
44. TWO TYPES OF PROTEIN
CONFORMATION
Fibrous proteins – collagen, fibroin (silk
fibers), and keratin (wool fibers)
Globular proteins – a more or less spherical
shape of proteins because of the back
folding of its backbone. Unlike fibrous
protein, it is water-soluble.
45. TERTIARY STRUCTURE OF PROTEIN
- 3-D arrangement of all the atoms in the
molecule
- Results due to folding, refolding, and
supercoiling of the polypeptide chain
leading to rod-like or globular structures
49. MYOGLOBIN
- Classical example of globular protein
- The first protein by which a complete
tertiary structure was determined by X-ray
Crystallography
- Consists of single polypeptide chain of 153
amino acid residues including the
prosthetic group, HEME group.
51. DENATURATION AND REFOLDING
Denaturation – unfolding of protein
(disruption of tertiary structure of protein)
1. Heat
2. Extremes of pH – high or low
3. Detergents – SDS, other reagents like
urea and guanidine HCl
4. β-mercaptoethanol – reduced disulfide
linkages
52. QUATERNARY STRUCTURE
- Consist of more than one polypeptide
chains (could be identical or different) or
subunits
- Dimers, trimers, tetramers (hemoglobin)
- Subunits are linked together by
noncovalent interactions such as
electrostatic interaction, H-bonds, and
hydrophobic interaction.
53. QUATERNARY STRUCTURE
- Due to noncovalent interactions, an
allosteric effect will occur
- Allosteric – subtle changes in structure on
a one site of protein that may cause
drastic changes in properties at a distant
site.
54. HEMOGLOBIN
- Tetramer (α2β2)
- Both α and β-chains are very similar to
hemoglobin structure
- α-chains consist of 141 amino acid residues
and β-chains consist of 146 amino acid while
hemoglobin has 153 amino acid residues
- Both α-chains and β-chains amino acids
residues have the same position as in
myoglobin, therefore they are HOMOLOGOUS
to each other
55. HEMOGLOBIN
- Myoglobin can only bind 1 molecule of
Oxygen while hemoglobin can bind 4
molecules
- Both binding are reversible however,
hemoglobin exhibits positive cooperativity
while myoglobin does not
Cooperative
binding
56. HOW DOES HEMOGLOBIN WORK?
Myoglobin – oxygen-storage in muscle. Bind
strongly to oxygen and it is 50% saturated
at 1 torr partial pressure of oxygen.
Hemoglobin – oxygen transport (binds
oxygen and releasing it depending on the
situation)
Alveoli of the lungs – 100% saturation
Capillaries of active muscles – 20 torr
(<50%)
58. CONFORMATIONAL CHANGES THAT
ACCOMPANY HEMOGLOBIN FUNCTION
Bohr effect – effect of H+ on conformation
of hemoglobin
CO2 concentration – increase the H+
concentration
59. CONFORMATIONAL CHANGES THAT
ACCOMPANY HEMOGLOBIN FUNCTION
Presence of 2,3-BPG – binding of BPG to
hemoglobin shows drastic effects on
oxygen-binding capacity
60. CONFORMATIONAL CHANGES THAT
ACCOMPANY HEMOGLOBIN FUNCTION
Presence of BPG resulted to the 50%
saturation of Oxygen at 26 torr. Without
BPG, oxygen binding would be greater than
50% and little oxygen would be release in
the capillaries
BPG also plays a role in supplying oxygen
to a growing fetus
61. PROPERTIES OF AMINO ACIDS
6. All amino acids except proline and
hydroxyproline react with nitrous acid
(HNO2), liberating nitrogen gas
7. Amino acids or a mixture of amino acids
react with formaldehyde and can be used as
an analytical method (formol titration) to
monitor the formation of free amino acids
during the hydrolysis of proteins by
proteolytic enzymes
62. PROPERTIES OF AMINO ACIDS
8. By heating with Ba(OH)2, primary amino
acids are formed due to the breaking of the
–COOH with the formation of CO2
9. When amino acids are dehydrated, they
unite with each other forming a ring of DKP
63. PROPERTIES OF AMINO ACIDS
10. Amino acids react with ninhydrin
(triketohydrindene) to yield CO2, ammonia,
and an aldehyde containing one less carbon
than the amino acids. The reaction yields a
blue or purple color.
64. PROPERTIES OF AMINO ACIDS
11.
Name of test Reagents Positive results Functional group
indicated
Biuret Test CuSO4, NaOH Light pink to violet
solution
Peptide linkage
Ninhydrin Test Ninhydrin Blue Solution* α-amino group (free)
Millon’s Test HgNO3 in HNO3 Red ppt. Phenol Group
Xanthoproteic
Reaction
Conc. HNO3, conc.
NaOH
Yellow Solution Aromatic Ring
Adamkiewicz Glacial HOAc, conc.
H2SO4
Purple ring Indole ring
Sakaguchi test α-naphthol in
NaOBr, NaOH
Red solution Guanido group
Reduced sulfur Pb(OAc)2, NaOH Black ppt Sulfhydryl group
65. PROTEIN PURIFICATION AND
CHARACTERIZATION TECHNIQUE
Separation and Purification of Proteins
1. Homogenization – breaking open the
cells.
a. Grinding using a blender – cells are
broken up and releasing soluble proteins
b. Sonicator – using sound energy to agitate
particles in a sample for extraction
c. Potter-Elvejhem homogenizer – thick wall
test tube in which a tight-fitting plunger
is passed
67. PROTEIN PURIFICATION AND
CHARACTERIZATION TECHNIQUE
3. Salting out – crude purification based on
solubility. Ammonium sulfate is added to
ppt. out the proteins from solution
4. Separation by Chromatography
68. CHROMATOGRAPHY
- Compounds can be separated according to
its affinity on the stationary phase
- Samples are carried out with a solvent
(mobile phase) and flows into the
stationary phase.
- Samples can therefore be separated since
components in the sample interacts with
the stationary phase at different extent.
69. CHROMATOGRAPHY
Column Chromatography - in which the
material that makes up the stationary
phase is packed with a column.
- the mobile phase (eluent) is passed
through a column (silica gel, alumina, or
cellulose)
70. TYPES OF CHROMATOGRAPHY
Size-exclusion chromatography – also called
gel-filtration chromatography. Separates
molecules on the basis of size.
- column is composed of cross-linked
gel particles (bead form), composed of
carbohydrate polymer such as dextran or
agarose or a polyacrylamide
71. TYPES OF CHROMATOGRAPHY
Affinity Chromatography – uses the specific
binding properties of many proteins
- column is made up of polymeric
material that is covalently liked to some
compound (ligand), that binds specifically to
the desired protein.
72. TYPES OF CHROMATOGRAPHY
Ion-Exchange Chromatography – similar to
affinity chromatography since both uses a
column resin that binds to the desired
protein.
- Ion-exchange resin has a ligand with a
positive charge or a negative charge
- Negatively charged resin – cation
exchanger
- Positively charged resin – anion exchanger
73. ELECTROPHORESIS
- Is based on the motion of the charged
particles in an electric field toward an
electrode of opposite charge
74. WHAT IS THE DIFFERENCE BETWEEN
AGAROSE GEL AND POLYACRYLAMIDE
GEL?
Agarose-based gels – often use in the
separation of nucleic acids
Polyacrylamide gel – for proteins
SDS-PAGE
Without SDS – native gel
75. ELECTROPHORESIS
Isoelectric focusing – differences in
isoelectric points in proteins because of the
differences in titratable groups
2-D gel electrophoresis – combination of
SDS-PAGE and Isoelectric focusing
