Biochemistry 3070

Amino Acids
&
Proteins

Biochemistry 3070 – Amino Acids & Proteins 1

 Biochemistry 3070

Learning Objectives:
1. Point out amino acids as building blocks of proteins;

2. Draw the general type of formula of amino acids;

3. Illustrate optical isomerism of amino acids;

4. Classify amino acids based on their R groups and

give interactions associated with R-groups;

5. Explain the acid-base behavior of amino acids and

their importance;

6. Calculate pH of amino acids and indicate the charge

of amino acids and proteins and explain the principle
behind the methods.
Biochemistry 3070 – Amino Acids & Proteins 2
 Biochemistry 3070

Outline:
A. Amino acid structure
B. Acid- base Properties of Amino acids
C. Classification of Amino acids
D. How amino acid are linked together

Biochemistry 3070 – Amino Acids & Proteins 3

 • Proteins are linear copolymers built from
monomeric units called amino acids - C,H,O,N
(S)
• Twenty amino acids are commonly found in
proteins also known as “STANDARD AMINO
ACID”
– Nonpolar
– Polar neutral
– Polar acidic
– Polar basic

Biochemistry 3070 – Amino Acids & Proteins 4

 • These amino acids contain a variety of different
functional groups:
– Alcohols (R-OH)
– Phenols (Ph-OH)
– Carboxylic acids (R-COOH)
– Thiols (R-SH)
– Amines (R-NH2)
– and others…

Biochemistry 3070 – Amino Acids & Proteins 5

 • Protein function depends on both
– amino acid content, and
– amino acid sequence.
• Protein fold into diverse shapes such as
– spherical
– elipsoidal
– long strands, etc.
• All information for 3-D structure is
contained in the linear sequence of amino
acids.

Biochemistry 3070 – Amino Acids & Proteins 6

Biochemistry 3070 – Amino Acids & Proteins 7
 • To understand protein function, we must first
understand the nature of amino acids.
• Amino acids are essentially α-amino acids:
alpha carbon (IUPAC #2 position)

H2N – C – COOH
|
R

• When R is not H, the alpha carbon is

asymmetric, giving rise to isomers.
Biochemistry 3070 – Amino Acids & Proteins 8
 • To understand protein function, we must first
understand the nature of amino acids.
• Amino acids are essentially α-amino acids:
alpha carbon (IUPAC #2 position)

H2N – C – COOH
|
R

• When R is not H, the alpha carbon

is asymmetric, giving rise to isomers.
Biochemistry 3070 – Amino Acids & Proteins 9
 • To understand protein function, we must first understand
the nature of amino acids.
• Amino acids are essentially α-amino acids:
alpha carbon (IUPAC #2 position)

H2N – C – COOH
|
R

• When R is not H, the alpha carbon is asymmetric,

giving rise to isomers.
• R group/ variable group – defines the chemical nature
Biochemistry 3070 – Amino Acids & Proteins 10
 Only L-amino acids are constituents of proteins.

“L” and “D” isomeric nomenclature is similar to the

“R” and “S” utilized in modern organic chemistry.
Biochemistry 3070 – Amino Acids & Proteins 11
 • Amino acids have an acidic group (-
COOH) and a basic group (-NH2) on the
same alpha-carbon atom.
• Amino acid are amphoteric

Biochemistry 3070 – Amino Acids & Proteins 12

 • Carboxylic acids are traditional Bronsted-
Lowery acids, donating a proton in
aqueous solution.
• The pKa for carboxylic acids is normally
around 2 to 5. That is, the pH at which
these acids are 50% ionized:

R-COOH  R-COO- + H+

pH= [less than 2]  [above 5]

Biochemistry 3070 – Amino Acids & Proteins 13

 • Amino groups function as bases,
accepting a proton.
• The pKa for amino groups is usually
around 9 – 10. Again, at the pKa these
groups are 50% ionized:

R-NH3+  R-NH2 + H+

pH= [below 8]  [above 9]

Biochemistry 3070 – Amino Acids & Proteins 14

 • Even though both acids and amines are present in the
same molecule, they mostly behave as though they were
separate entities:

Biochemistry 3070 – Amino Acids & Proteins 15

Biochemistry 3070 – Amino Acids & Proteins 16
 • Summary:
At low pH, proton concentration [H+]is high.
Therefore, both amines and carboxylic
acids are protonated. (-NH3+ & -COOH)
At high pH, proton concentration is low.
Therefore, both amines and carboxylic
acids are deprotonated. (-NH2 & -COO-)
At neutral pH, amines are protonated(-NH3+)
and carboxylates are deprotonated(-COO-)

Biochemistry 3070 – Amino Acids & Proteins 17

 • “Zwitter” Ions:
• A molecule having a positive charge on an
atom, a negative charge on a different
atom, and no net charge.

Amino acid occur as neutral

on both solid and in neutral

solutions.
Biochemistry 3070 – Amino Acids & Proteins 18
 • “Zwitter” Ions:
• Ions bearing two charges were named
zwitter ions by German scientists; the
name still applies today, especially for
amino acids at neutral pH:

-
+
H3N – CH2 – COO

Biochemistry 3070 – Amino Acids & Proteins 19

 Acid-Base Properties of Amino Acids
Draw the following chemical structures for glycine:
(Non-existent form:) H2N – CH2 - COOH

pH=1:

pH=7:

pH=12:

Biochemistry 3070 – Amino Acids & Proteins 20

 Acid-Base Properties of Amino Acids
Draw the following chemical structures for glycine:
(Non-existent form:) H2N – CH2 - COOH

pH=1: H3N – CH2 - COOH

+

pH=7:

pH=12:

Biochemistry 3070 – Amino Acids & Proteins 21

 Acid-Base Properties of Amino Acids
Draw the following chemical structures for glycine:
(Non-existent form:) H2N – CH2 - COOH

pH=1: H3N – CH2 - COOH

+

pH=7: H3N – CH2 – COO-

+

pH=12:

Biochemistry 3070 – Amino Acids & Proteins 22

 Acid-Base Properties of Amino Acids
Draw the following chemical structures for glycine:
(Non-existent form:) H2N – CH2 - COOH

pH=1: H3N – CH2 - COOH

+

pH=7: H3N – CH2 – COO-

+

pH=12: H2N – CH2 – COO-

Biochemistry 3070 – Amino Acids & Proteins 23

 Low pH Neutral pH High pH

Three form exist:

Biochemistry 3070 – Amino Acids & Proteins 24

 SEATWORK NO. 1
Draw the following chemical structures for valine:
(Non-existent form:) H2N – CH (CHCH3CH3)- COOH

pH=1:

pH=7:

pH=12:

Biochemistry 3070 – Amino Acids & Proteins 25

 Amino acids: (Nonpolar - Aliphatic)

Biochemistry 3070 – Amino Acids & Proteins 26

 • Amino acids (Nonpolar - Aromatic)

Biochemistry 3070 – Amino Acids & Proteins 27

 • Amino acid Proline
(The only secondary (2°) amino acid or (“imino” acid.)

Biochemistry 3070 – Amino Acids & Proteins 28

 • Amino acids (Polar - Alcohols)

Biochemistry 3070 – Amino Acids & Proteins 29

 • Amino acids (Sulfur)

cystine

Biochemistry 3070 – Amino Acids & Proteins 30

 • Amino acids (Polar: Acids and related
amides)

Biochemistry 3070 – Amino Acids & Proteins 31

 • Amino acids (Polar: Basic)

Biochemistry 3070 – Amino Acids & Proteins 32

 • Histidine (Acid/Base Activity)

Biochemistry 3070 – Amino Acids & Proteins 33

Biochemistry 3070 – Amino Acids & Proteins 34
Biochemistry 3070 – Amino Acids & Proteins 35
 Essential Amino Acids: Codes (three-/one-letter)
R
Arginine b Arg
H
Histidine b His
Isoleucine Ile I
Leucine Leu L
Lysine Lys K
Methionine Met M
F
Phenylalanine a Phe
Threonine Thr T
W
Tryptophan a Trp
Valine Val V
a
Aromatic b
Probably essential

Biochemistry 3070 – Amino Acids & Proteins 36

 The two types of protein-energy
malnutrition

Biochemistry 3070 – Amino Acids & Proteins 37

 The two types of protein-energy
malnutrition

• Kwashiorkor is a Ghanaian
word that means "the disease that
the first child gets when the new
child comes"
• characteristic symptom
• = swollen abdomen
• Energy intake could be adequate,
but protein consumption is too low.

Biochemistry 3070 – Amino Acids & Proteins 38

 The two types of protein-energy
malnutrition

• Marasmus means "to waste

away" or "dying away", and thus
occurs in individuals who have
severely limited energy intakes.

Biochemistry 3070 – Amino Acids & Proteins 39

 The two types of protein-energy
malnutrition

Biochemistry 3070 – Amino Acids & Proteins 40

 • Amino acids are polymerized via amide or
“peptide” bonds:

Biochemistry 3070 – Amino Acids & Proteins 41

 • Copolymer of amino acids:
– a “polypeptide”

Definition:
Amino acid polymers of ≤50 amino acids are called
“polypeptides, peptides, oligopeptides, etc.”
Amino acids polymer of >50 amino acids are called “proteins.”

Biochemistry 3070 – Amino Acids & Proteins 42

 • PEPTIDE:
• Dipeptide
• Tripeptide
• oligopeptides (10-20)
• Polypeptide (long unbranched chain of aa)

Biochemistry 3070 – Amino Acids & Proteins 43

 N-terminal backbone C-terminal
Biochemistry 3070 – Amino Acids & Proteins 44
 • An example of a “dipeptide” is the sweetener
Aspartame.
• Other names include:
– NutraSweet
– Equal
– Tri-Sweet
– Sanecta
• IUPAC Name:
“N-L- α – Aspartyl-L-phenylalanine 1-methyl ester”

Abbreviated Structure:
Asp – Phe - OCH3

Biochemistry 3070 – Amino Acids & Proteins 45

 • Cross links between peptide chains:
– Disulfide linkages between individual
“cysteines” are called “cystines:”

Biochemistry 3070 – Amino Acids & Proteins 46

 • Insulin is the smallest protein, with 51
amino acids in two chains linked by
cystine (disulfide) cross links:

Biochemistry 3070 – Amino Acids & Proteins 47

 Glucagon
• Although the injection of insulin lowers the blood sugar,
administration of glucagon, another pancreas hormone,
raises the blood sugar level.
• Glucagon - straight peptide chain of 29 amino acids.
It has been synthesized; the synthetic product has the
full biological activity of natural glucagon. The structure
of glucagon is free of cystine and isoleucine.

Biochemistry 3070 – Amino Acids & Proteins 48

 Glucagon
• Although the injection of insulin lowers the blood sugar,
administration of glucagon, another pancreas hormone,
raises the blood sugar level.
• Glucagon - straight peptide chain of 29 amino acids.
It has been synthesized; the synthetic product has the
full biological activity of natural glucagon. The structure
of glucagon is free of cystine and isoleucine.

Biochemistry 3070 – Amino Acids & Proteins 49

 Immunoglobulins
• Antibodies, proteins that combat foreign
substances in the body, are associated
with the globulin fraction of the immune
serum.
• serum globulins are separated into α-, β-,
and γ- fractions, antibodies are associated
with the γ-globulins aka immunoglobulins.

Biochemistry 3070 – Amino Acids & Proteins 50

 Immunoglobulins Superfamily
• Characteristics:

Biochemistry 3070 – Amino Acids & Proteins 51

 Immunoglobulins Superfamily
• Characteristics:

Biochemistry 3070 – Amino Acids & Proteins 52

 Immunoglobulins Superfamily
• Characteristics:

Biochemistry 3070 – Amino Acids & Proteins 53

 Immunoglobulins Superfamily
• Characteristics:

Biochemistry 3070 – Amino Acids & Proteins 54

 Hormone
• Thyrotropin-releasing Hormone
– Secreted by hypothalamus; causes anterior
pituitary gland to release thyrotropic hormone

Biochemistry 3070 – Amino Acids & Proteins 55

 Hormone
• Thyrotropin-releasing Hormone
– Secreted by hypothalamus; causes anterior
pituitary gland to release thyrotropic hormone

• Vasopressin (antidiuretic hormone)

– Secreted by posterior pituitary gland; causes
kidney to retain water from urine

Biochemistry 3070 – Amino Acids & Proteins 56

 • Peptide bonds have partial double bond
character due to resonance that limits
rotation about this bond:

Biochemistry 3070 – Amino Acids & Proteins 57

Biochemistry 3070 – Amino Acids & Proteins 58
 Levels of Protein Structure

• Primary (1°) Protein Structure

– linear sequence of amino acids.
• Secondary (2°) Protein Structure
– localized regional structures
• Tertiary (3°) Protein Structure
– overall shape of proteins
• Quaternary (4°) Protein Structure
– interactions between proteins

Biochemistry 3070 – Amino Acids & Proteins 59

 Levels of Protein Structure

• Primary (1°) Protein Structure

– the order in which the individual amino acids making
up the protein are linked together through peptide
bonds

Biochemistry 3070 – Amino Acids & Proteins 60

 Levels of Protein Structure

• Primary (1°) Protein Structure

– the order in which the individual amino acids making
up the protein are linked together through peptide
bonds
– location of disulfide (cystine) bonds.

Biochemistry 3070 – Amino Acids & Proteins 61

 Levels of Protein Structure

• Primary (1°) Protein Structure

– the order in which the individual amino acids making
up the protein are linked together through peptide
bonds
– location of disulfide (cystine) bonds.
– understand its structure and mechanism of action

Biochemistry 3070 – Amino Acids & Proteins 62

Biochemistry 3070 – Amino Acids & Proteins 63
Biochemistry 3070 – Amino Acids & Proteins 64
 Levels of Protein Structure

• Primary (1°) Protein Structure

– the order in which the individual amino acids making
up the protein are linked together through peptide
bonds

Biochemistry 3070 – Amino Acids & Proteins 65

 Protein Structure:

• Twisting about various bonds in the

polypeptide backbone gives proteins a
variety of shapes.

• Bond angles give rise to secondary

structures. Then, localized secondary
structures help drive the peptide folding
that gives rise to tertiary structure.

Biochemistry 3070 – Amino Acids & Proteins 66

 Secondary Structure in Proteins:
• Pauling and Corey proposed two
secondary structures in proteins many
years before they were actually proven:
alpha – helix beta - sheet

Both of these secondary protein structures

are stabilized by hydrogen bonding between
the carbonyl oxygen atoms and the nitrogen
atoms of amino acids in the protein chain.
Biochemistry 3070 – Amino Acids & Proteins 67
 • The alpha (α) – helix:

Biochemistry 3070 – Amino Acids & Proteins 68

 • The alpha (α) – helix:

Keratin

Biochemistry 3070 – Amino Acids & Proteins 69

 • beta – sheet (antiparallel):

Biochemistry 3070 – Amino Acids & Proteins 70

 • beta – sheet (artistic representations):

Biochemistry 3070 – Amino Acids & Proteins 71

 Examples of beta-sheet domains in proteins:

Biochemistry 3070 – Amino Acids & Proteins 72

Biochemistry 3070 – Amino Acids & Proteins 73
 • Tertiary (3°) Structure of Protein
Water-soluble proteins fold into compact structures with nonpolar cores.

Biochemistry 3070 – Amino Acids & Proteins 74

 Tertiary (3°) Structure the Protein Myoglobin
Water-soluble proteins fold into compact structures with non-polar cores.
consists of eight alpha helices connected by loops; contains 154 amino
acids and a porphyrin ring with an iron at its center.

Biochemistry 3070 – Amino Acids & Proteins 75

 • In the case of myoglobin and many other
proteins, the majority of hydrophobic amino
acids (yellow) are found inside in structure:

Biochemistry 3070 – Amino Acids & Proteins 76

 • The Cro protein of Lambda bacteriophage
is a dimer of identical subunits:

Biochemistry 3070 – Amino Acids & Proteins 77

 • Hemoglobin is a protein tetramer,
containing two identical pairs of subunits:

Biochemistry 3070 – Amino Acids & Proteins 78

 • The coat of rhinovirus contains 60 copies
of each of four subunits (240 total)!

Biochemistry 3070 – Amino Acids & Proteins 79

 He also used dialysis to separate these chemicals
from the enzyme in different orders.

Biochemistry 3070 – Amino Acids & Proteins 80

 Protein denaturation
The partial or complete disorganization of a proteins
characteristic three-dimensional shape as a result of
disruption of its secondary, tertiary, and quaternary
structural interactions.

Biochemistry 3070 – Amino Acids & Proteins 81

 Protein denaturation

Partial/complete Lose of function

Reversible/irreversible
Example:
Cooking
Sealing of blood vessels – cauterization
Temperature above 41⁰C (lethal to body)
Applying isopropyl alcohol or ethyl alcohol (70%)
Waving hair or strengthening (rebonding)

Biochemistry 3070 – Amino Acids & Proteins 82

 Protein denaturation
Physical and Chemical Denaturing Agents

Biochemistry 3070 – Amino Acids & Proteins 83

PROTEINS SEPARATION & TECHNIQUES
LEARNING OBJECTIVES:
 APPRECIATE THE IMPORTANCE OF PROTEIN
SEPARATION AND PURIFICATION
 IDENTIFY THE PROCESS OF PROTEIN
PURIFICATION AND SEPARATION
 DETERMINE THE TECHNIQUES OF PROTEIN
SEPARATION BASED ON:
CHARGED
SIZE/MOLECULAR MASS
SOLUBILITY
AFFINITY
 IDENTIFY THE PRINCIPLE BEHIND EVERY
TECHNIQUES OF PROTEIN SEPARATION AND
Protein sample
Charge (CICIE)
Molecular size
(MuM)
Binding affinity
(BAC)
Solubility (SOS)
Protein sample

Charge (CICIE) I solelectric focusing

C
Molecular size
(MuM) I
Binding affinity E
(BAC)
Solubility (SOS)
Protein sample

Charge (CICIE) I solelectric focusing

Capillary Electrophoresis
Molecular size
(MuM) I
Binding affinity E
(BAC)
Solubility (SOS)
Protein sample

Charge (CICIE) I solelectric focusing

Capillary Electrophoresis
Molecular size
(MuM) IOn-exchange column
chromatography
Binding affinity E
(BAC)
Solubility (SOS)
Protein sample

Charge (CICIE) I solelectric focusing

Capillary Electrophoresis
Molecular size
(MuM) IOn-exchange column
chromatography
Binding affinity Electrophoresis
(BAC)
Solubility (SOS)
A. BASED ON CHARGE (CICIE)

Isoelectric
Focusing
proteins are separated
according to their
isoelectric points
A. BASED ON CHARGE (CICIE)

Capillary Electrophoresis
Proteins dissolved in buffer
solution at particular pH placed in
electric field

Depending on the relationship of

buffer pH to pI, proteins moves

Cathode
Anode
Stationary (pH = pI)
A. BASED ON CHARGE (CICIE)
silica

Capillary Electrophoresis
Proteins dissolved in buffer
solution at particular pH placed in
electric field

Depending on the relationship of

buffer pH to pI, proteins moves

Cathode
Anode
Stationary (pH = pI)

ADVANTAGE:high separation efficiency

Use very small samples
Requires only several minutes for assay of samples
A. BASED ON CHARGE (CICIE)

Capillary Electrophoresis
A. BASED ON CHARGE (CICIE)

Ion-exchange Column Chromatography

Proteins are separated by charged ion
exchange resin
Negatively charged resin – cation-exchange
resin
bind to (+) charged ligand
ex. carboxymethyl
Positively charged resin – anion-exchange resin
bind to (-) charged ligand
ex. Diethylamino group
Released by adjusting the pH buffer or salt
concentration of the elution buffer
A. BASED ON CHARGE (CICIE)

Electrophoresis Proteins dissolved in buffer

Method of separating a mixture of amino acids, proteins
and DNA
solution at particular pH placed in
electric field
Depending on the relationship of
buffer pH to pI, proteins moves

Cathode
Anode
Stationary (pH = pI)
Polymers :
Polyacrylamide
agarose
cellulose acetate
Protein sample
Charge (CICIE)
Molecular size Ultracentrifugation
(MuM)
Binding affinity
(BAC)
Solubility (SOS)
Protein sample
Charge (CICIE)
Molecular size Ultracentrifugation
(MuM) MOlecular exclusion
Binding affinity
(BAC)
Solubility (SOS)
B. BASED ON MOLECULAR MASS/SIZE (MUM)

Ultracentrifugation
An equipment in
which protein is
subjected to
centrifugal force
moves in the direction
of force at a velocity
dependent on its mass
B. BASED ON MOLECULAR MASS/SIZE (MUM)

Molecular Exclusion
Chromatography
a porous gel (insol. Beads) used to
separate proteins by size

Smaller proteins penetrate pores of

gel and between beads
larger proteins between beads only
and flow more rapidly in the column
Protein sample

Charge (CICIE)
Molecular size
(MuM)
Binding affinity Affinity Chromatography
(BAC)
Solubility (SOS)
Protein sample

Charge (CICIE)
Molecular size
(MuM)
Binding affinity Affinity Chromatography
(BAC) Column Chromatography
Solubility (SOS)
C. BASED ON BINDING AFFINITY (BAC)

Affinity Chromatography
High affinity compounds
covalently linked or attached to an
insoluble resin = modified resin

Thus when a mixture of proteins

containing the protein that
recognizes the ligand is applied to
the column, other proteins pass
through in a wash of the column,
while the protein of interest is
retained.
 Column Chromatography

Molecular exclusion - size

Ion-exchange – net charge at a given pH
Affinity – ligand binding specificity
Protein sample

Charge (CICIE)
Molecular size
(MuM)
Binding affinity
(BAC)
S Alting out
Solubility (SOS)
D. BASED ON SOLUBILITY (SOS)
Salting out
E. OTHERS
High Performance Liquid Chromatography
Liquid solvent containing the
mixture of molecules to be
identified is passed through
column densely packed with small
diameter insoluble beadlike resin

Smaller-tightly packed=greater
resolution

Uses high pressure pumps with

metal plumbing
E. OTHERS
High Performance Liquid Chromatography
E. OTHERS
Two-Dimensional (2D) Gel Electrophoresis
Protein extracted from cell/tissue, Intensity of each spot is measured
spotted in electrophoresis gel in determine type and concentration
electrophoresis apparatus
Spot may be extracted from gel and
Separated on differences on pI hydrolyzed peptide fragments
Gel is turned by 90 degrees and
SDS is added Peptide fragments is subjected to
Protein separated on basis of mass spectroscopy
differences in molecular mass
Resulting gel is stained for proteins
E. OTHERS
Two-Dimensional (2D) Gel Electrophoresis
Horizontal separation of spots is
based on their pI differences.
Vertical separation is based on
differences in molecular weights.
 Thank you for your attention!

Biochemistry 3070 – Amino Acids & Proteins 111