Amino Acids and

Proteins
 AMINO
ACIDS
• molecules containing an amine group, a carboxylic acid group and a
side chain that varies between different amino acids.
• Common/Standard amino acids
- 19 α-amino acids
- 1 heterocyclic amino acid (imino acid)

• Uncommon/Rare amino acids

-produced by post-translational
modification of the common amino acid (i.e.
hydroxyproline, hydroxylysine, and thyroxine)
 AMINO
ACIDS
What is an amino acid ?

~ In chemistry : A molecule that consists of both amine

and carboxyl functional groups

~ In biochem : Amino acid is known as alpha amino

acid, where the amino and carboxylic
acid group are attached to the same
carbon (α–carbon).
 Optical Properties of the Amino Acids
~ A tetrahedral carbon atom with 4 distinct constituents is said to
be chiral.
~ able to rotate the plane of polarized light either to the right
(dextrorotatory) or to the left (levorotatory).
~ All amino acids in proteins exhibit the same absolute steric
configuration as L-glyceraldehyde. Therefore, they are all L-
alpha-amino acids.
~ D-amino acids are never found in proteins, only found in
polypetide antibiotics.
~ Example : optical isomers of alanine
Amino Acid Classifications
~ There are two broad classes of amino acids based upon whether the
R-group is hydrophobic or hydrophilic.
a) Hydrophobic : tends to repel the aqueous environment
(does not ionize or form H-bonds)
b) Hydrophilic : tends to interact with the aqueous
environment
(often involved in the formation of H-bonds)

Example :
Chemical Nature of Amino Acids
~ The various alpha amino acids differ in the side chain that is
attached to their alpha carbon.
~ Examples of side chain :
a) both hydrogen : glycine

alanine
b) methyl group :

tryptophan
c) heterocyclic :
group
~ All peptides and polypeptides are polymers of alpha-amino acids.
~ There are 20 alpha-amino acids that are relevant to the make-up of
mammalian proteins.
 Physical properties
a) Optically active
b) Soluble in water
formation of hydrogen bond
c) High boiling point
d) High melting point – formation of lattice structure
in solid state
e) Forms zwitterions in aqueous state

R1 R1
H2 O
+ -
H2 N C COOH H3 N C COO

R2 R2
 Chemical reactions
a) acid base reactions
b) condensation polymerisation
c) hydrolysis

 Acid base reactions

~ not in aqueous :
(1) HOOCCH2NH2 + H+ HOOCCH2NH3+
(2) HOOCCH2NH2 + OH- -
OOCCH2NH2

~ in aqueous :
(1) -OOCCH2NH3+ + H+ HOOCCH2NH3+
(2) -OOCCH2NH3+ + OH- -
OOCCH2NH2 + H2O
Condensation polymerisation
~ definition : In condensation polymerization, monomers join
to each other producing a small molecule as a byproduct. The
small molecule is often water.

~ Proteins are created by condensation polymerisation of amino

acids which yields the newly formed peptide bond and a water
molecule.

two
dipeptide polypeptide protein
amino acids
 Condensation polymerisation

amino acid + amino acid dipeptide + water

 The peptide bond
~ Peptide bond formation is a condensation reaction leading to the
polymerization of amino acids into peptides and proteins.
~ The presence of the carbonyl group in a peptide bond allows
electron resonance stabilization to occur such that the peptide bond
exhibits rigidity not unlike the typical -C=C- double bond.
~ The peptide bond is, therefore, said to have partial double-bond
character.

Resonance stabilization forms of the peptide bond

Summary of the peptide bond formation

~ The simplest peptide, a dipeptide, contains a single peptide bond

formed by the condensation of the carboxyl group of one amino acid with
the amino group of the second with the elimination of water.
~ Peptides are small, consisting of a few amino acids.
~ Proteins are polypeptides of greatly divergent length.
Polypeptide ( Mr > 10000)
H R1 O H R1 O

N C C N C C

n
R2 R2
 Protein
~ condensation polymers formed from amino acid monomers
~ layers of polypeptides bound by hydrogen bond

H-bond
  Hydrolysis
H R1 O H R1 O
N C C N C C
H R2 R2 OH

dipeptide

+ H2 O

R1 R1

O H O
H
N C C + N C C
H OH H OH

R2 R2

amino acid amino acid

** Rate of reaction is very slow : use acidic or alkaline hydrolysis

STRUCTURE OF COMMON AMINO
ACIDS
(Hydrophobic) Basic)

(Uncharged) (Acidic)
Amino
Acids
Amino
Acids
Amino
Acids

Nelson and Cox, 2004

GENERAL PROPERTIES OF COMMON AMINO ACIDS

• Crystalline solids at room temperature

• Polar - generally soluble in water which is attributed to –OH and –NH2

- interact with water by H-bonding in reciprocal manner but differ in
degree

• Strong dipoles – characterized by high dipole moment

• Characterized by high melting points (implies strong interaction

between molecules by H-bonding)
GENERAL PROPERTIES OF COMMON AMINO ACIDS

• Electrolytic – poor conductors of electricity

• Stereochemical properties – exhibit configurational stereoisomerism

Stereocenter: chiral or assymetric C-atom

for all common a.a except Gly = α-carbon

for Ile & Thr = α and β-carbon
GENERAL PROPERTIES OF COMMON AMINO ACIDS

• Stereochemical properties
– exhibit optical isomerism; L (-) or D (+) except Gly
- exhibit diastereoisomerism (for Ile and Leu)

• Amphoteric
– exhibits acid-base properties
- acid property due to carboxylic acid functional group (Arrhenius)
- basic property due to the amino group (Lewis)
GENERAL PROPERTIES OF COMMON
AMINO ACIDS
• Amphoteric
– exists as a zwitterion or dipolar ion at isoelectric pH (pI)

- The isoelectronic point or

isoionic point is the pH at which
the amino acid does not migrate
in an electric field. This means
it is the pH at which the amino
acid is neutral
ESSENTIAL AMINO ACIDS

Val Thr Met Arg

Phe
PVT TIM HALL Leu

His Lys
Trp Ile

• These amino acids must be provided in the diet since they cannot be biosynthesized (Arg
and His are not sufficiently biosynthesized).

• Inadequate supply of any of these a.a. leads to poor growth and failure to thrive
PROTEINS
PROTEINS
• Most abundant biomolecule in all cells and all parts of a
cell

• Biopolymer of amino acids

• 20 common amino acids

• Wide range of structure & functions

Protein Amino Acid
◦ Protein diversity – is based on different
arrangements of a common set of 20 amino acid
monomers
◦ A combination of different amino acids will
produce different types of proteins ( structure )
which have different functions .
PROTEI
NS
• Also known as polypeptides

• organic compounds made of amino acids arranged in a linear chain.

• The amino acids in a polymer are

joined together by the peptide
bonds between the carboxyl and
the amino groups of adjacent
amino acid residues.
◦ The making of a protein chain involves different amino acids which
can be repeated but they must have a proper order.

◦ Different protein – different length

Insulin - 51 Amino Acids
Haemoglobin – 500+ Amino Acids
Keratin – Thousands Amino Acids
◦ Amino Acids are commonly classified into the following groups based on the
chemical and/or structural properties of their side chains
◦ For example, amino acids with

1. polar ( hydrophilic) side chains will be called ‘Polar Amino

Acids’
2. non polar ( hydrophobic ) side chains will be called ‘Non
Polar Amino Acids’
3. electrically charged amino acids will be called ‘Electrically
charged amino acids’
Amino acid classifications
Protein Shape Function
◦ A protein consists of polypeptide chains folded into a unique shape
◦ The shape determines the protein’s function
◦ A protein loses its specific function when its
polypeptides unravel
◦ Each sequence of amino acid spontaneously folds in a different way.
◦ The folding creates groove that function as a binding site for other molecules.
◦ Changes in pH, heat or salinity can cause protein to unfold and lose their functionality – denaturation.
Amino Acid Sequence
◦ The order of amino acids as they occur in a polypeptide chain = chain of amino acids
◦ This is referred to as the primary structure of proteins.
◦ It is of fundamental importance in determining protein conformation.

Amino acid
sequence in beef
insulin
How does an amino acid links to another to
form a peptide then into protein?

• Peptide bond
• Condensation
GENERAL PROPERTIES OF
PROTEINS
• Water Solubility
– depends on the polarity of the R-group of the predominant a.a. residues.
- More a.a. with polar/ionic R-groups = more soluble in water
- soluble proteins are colloidally dispersed in water

• Polyampholytes
- polymers which are amphoteric electrolytes
- Their aqueous solutions are poor conductors of electricity
GENERAL PROPERTIES OF PROTEINS
• Polyampholytes
- Exhibits acid-base property:
a. In acid solutions: proteins are (+) charged forming protein salts
b. In basic solutions: proteins are (-) charged forming proteinates
- Each protein has its own isoelectric pH (pI)
If pH < pI proteins are positively charged)
, pH > pI proteins are negatively charged)
pH = pI proteins are zwitterionic / dipolar ionic

When pH of the medium is equal to the pI of the protein, the protein becomes least soluble
and can be precipitated (Isoelectric precipitation)
PROTEIN STRUCTURES
Levels of Protein Structure
 Primary Structure
 sequence of amino acid residues that serves as the backbone of a
peptide chain or protein
 function of the protein depends on the 1o structure
 Secondary Structure
 the “local” ordered structure brought about via H-bonding mainly
within the backbone
 α-helix & β-pleated sheets
 Levels of Protein Structure
 Secondary Structure - α-helix
 Rod-like
structure

 N-H to C=O
H-bonds in 4th
succeeding
amino acids

 H-bonds
parallel to axis

 Typically
amphiphilic
Levels of Protein Structure
 Secondary Structure – β-pleated sheets
 Polypeptide chains are arranged  Parallel or antiparallel
side by side  More stable
 H-bonds form between chains
Levels of Protein Structure
 Tertiary Structure
 Specific overall shape of a protein
 Cross links between R-groups of amino acids in chain
 Disulfide ----S—S—
 Ionic ---COO-------
 H-bonds H3N+---
 Hydrophobic
Levels of Protein Structure
 Quaternary Structure
 Aggregates of two or more protein chains connected by
weak
non-covalent interactions
 Proteins with two or more chains
 Example: Hemoglobin
 Carries O2 in blood
 Four polypeptide chains
Each chain has a heme
group
PROTEIN
CLASSIFICATION
Classification of Proteins Accdg. To
Composition
1. SIMPLE PROTEINS
- yield only amino acids upon
hydrolysis

2. CONJUGATED PTOTEINS
simple proteins + non-protein
substances ex. Nucleoprotein
Glycoprotein
Lipoprotein
Phosphoprotein
Hemoprotein
Metalloprotein
 Accdg. To Biological
Function
1. ENZYMES
2. TRANSPORT PROTEINS
3. NUTRIENT and STORAGE PROTEINS
4. STRUCTURAL PROTEINS
5. CONTRACTILE and MOTILE PROTEINS
6. DEFENSE PROTEINS
7. REGULATORY PROTEINS
Accdg. To Biological
Function
• Catalytic enzymes
• Transport hemoglobin, transferrin, albumin
• Storage ovalbumin, casein, gliadin
• Contraction actin, myosin
• Gene regulation histones, non-histone nuclear
• Regulatory hormones, repressor proteins
• Immune protection antibodies, fibrin, complement
• Structural collagen, elastin, keratin
• Neurotransmission receptor proteins
 Accdg. to
Shape
1. GLOBULAR PROTEINS
-polypeptide chain/s folded into
spherical or globular shape
-soluble in aq. system
-ex. Enzymes

2. FIBROUS PROTEINS
-polypeptide chains arranged in long
strands or sheets
-water insoluble
-ex, keratin, collagen, fibroin
 Accdg. To
Solubility
1. ALBUMINS (plasma)
- soluble in water and dilute aq. solutions
2. GLOBULINS (serum)
- soluble in dilute salt solutions but are insoluble
or sparingly soluble in water
3. GLUTELINS (flour)
- soluble in dilute solutions of acids and bases
- insoluble in neutral solvents
 Accdg. To
Solubility
4. PROLAMINS (store protein in plants)
- soluble in 50-90% alcohol
- insoluble in water, neutral solvents or absolute
alcohol
5. ALBUMINOIDS/SCLEROPROTEINS
- albumin-like that is insoluble in in most ordinary
solvents
 Protein Denaturation
A loss of three-dimensional structure sufficient to
cause of loss of function.

Types:
1. Irreversible Denaturation
- biological function /activity
cannot be regained
2. Reversible Denaturation
- biological function/activity can be
regained
 Denaturating
Agents
Proteins can be denatured by:
a. Strong acids and bases
b. Organic solvents
c. Detergents
d. Reducing agents
e. Salts
Salting-in
Salting-out
f. Heavy
metals
g. Temperatur
 Protein
Hydrolysis
1. COMPLETE HYDROLYSIS
- uses a strong acid or base +
high T
- product/s: amino acids

2. INCOMPLETE/PARTIAL HYDROLYSIS
- uses enzymes called protease
- product/s: mixture of amino acids and
oligopeptides
 COMPLETE
HYDROLYSIS
1. ACID HYDROLYSIS
- most commonly used reagent is 6N HCl
-disadvantages:
a. partial destruction of cys and tyr
b. complete destruction of trp
c. incomplete liberation of val and ile
d. racemization and destruction of ser and thr
e. asn + gln converted to asp + glu
 COMPLETE
HYDROLYSIS
2. ALKALINE HYDROLYSIS
- uses NaOH or KOH
-advantages:
a. trp not
destroyed
-disadvantages:
a. arg, asn, gln,
ser are
destroyed
 Partial
Hydrolysis
Catalysts (eg. acid, base, enzyme) are needed to facilitate the hydrolysis of
peptide bonds.

Enzymatic Hydrolysis - presence of proteolytic enzymes results to

partial or selective hydrolysis of polypeptide to yield a mixture of peptide
fragments.

Proteases/Peptidases – are enzymes that hydrolyze peptide

bonds at specific sites

Advantages: Amino acids are not affected; it requires certain

temperature & pH conditions for optimum activity of enzymes.
Enzymatic Hydrolysis

 Exopeptidases
a) Carboxypeptidase A – C-terminal residues except R,K and P
b) Carboxypeptidase B – R or K at C-terminus
c) Aminopeptidase – most N-terminal except when P is the next residue
 Endopeptidases
a) Trypsin – C side R & K
b) Chymotrypsin – C side of F, Y and W
c) Papain – C side of hydrophobic groups/ aromatic-R groups
 Separation/Purification of
Proteins
Properties of proteins being considered:
1. Charge
2. Molecular size, shape
3. Solubility
4. Affinity to a ligand
5. pI/IpH
EXPERIMENT
PROTEINS
Isolation, Hydrolysis and
Characterization
Isolation of Casein and Albumin
from
Cow’s Milk
 Casein
• Source: Skimmed milk
• Isolation method: Isoelectric precipitation
• A phosphoprotein (Phosphate groups attached to OH
groups of ser or thr); calcium caseinate
• present as micelles in milk
• Acts as a storage for amino acids
• Its isoelectric point is at pH 4.6

Ca2+Caseinate + 2 HCl → Casein + CaCl2

 Casein
• It is a conjugated protein (phosphoprotein). PO4—3
interacts (ionic) with Ca+2 leading to polymerization of
casein:
[casein – P – Ca+2 – P – casein]
Albumin
• Source: Skimmed milk
• Isolation method: Denaturation and Coagulation by heat
• Globular proteins that are soluble in water and diluted salt
solutions
• Second major protein in bovine milk
• Metalloprotein that can bind to several metal ions like
calcium and zinc
• It can serve as a regulatory protein in lactose
biosynthesis
 Myoglobin
• Source: Beef muscle
• Isolation method: Salt-induced precipitation
 Myoglobin
• Small, bright red protein common in muscle cells
• Stores oxygen (used when muscles are hard at work)
• A hemoprotein containing a heme group at its center
• Principle involved in isolation:
• Salting in followed by salting out
• Salting in - myoglobin is released in the cell & is soluble at 70% (NH4)2SO4
solution
• Salting out- increase ionic strength by addition of more (NH4)2SO4 resulting to
decrease solubility of proteins (due to competition between salt ions and
proteins, with water preferring to dissolve the salt rather than the proteins.
• Isolated myoglobin is a white precipitate.
 Gluten
• Source: Wheat flour
• Isolation method: Solubility difference
• Wheat, rye, and barley
• Gives bread its structure, texture and elasticity
• It is a composite of a prolamin and a glutelin, which
exist, conjoined with starch
• Storage protein responsible for
the elasticity and extensibility
of dough
 Gluten
• Consists of gliadin and glutenin

• Isolated gluten free of starch when (-) to iodine test

• Gluten are long molecules that are strong and flexible.

• Role of gluten in bread-making: it traps the CO2 produced by

the reaction of flour & yeast and gives flour its characteristic
chewiness.
 Gluten
• Principle involved in isolation:
• Difference in solubility – starch is partially soluble in H2O
while gluten is insoluble in H2O.

• I2 solution is used to test the complete removal of

starch.

• It is a yellowish-white solid, tough, elastic, and

sticky.
CHEMICAL TESTS FOR
PROTEINS
 Qualitative Color
Reactions
These are test reactions that qualitatively identify specific
functional group present in the solution of protein of
interest.

-- Biuret test -- Fohl’s test

-- Ninhydrin test
-- Xanthoproteic test
-- Millon’s test
-- Hopkins Cole test
-- Sakaguchi test
-- Nitroprusside test
-- Test for amide
-- Pauly’s test
 Biuret test
 Purpose: Used to detect the presence of peptide bonds
 Reagents: NaOH & CuSO4
 (+) result: pink to violet to blue coloration
 Principle: Complexation of Cu2+ with a peptide bond
 Ninhydrin test
 Purpose: detects free α-amino acids & amines
Reagents: 1,2,3-indanetrione monohydrate/ triketohydrindene &
EtOH
(+) result: blue → blue-purple color or yellow-orange product
(Proline)
Principle: oxidative decarboxylation & deamination followed by
condensation

Ninhydrin Fingerprinting
 Xanthoproteic test
 Purpose: detects aromatic amino acids (Y, W & F)
 Reagents: conc. HNO3 & conc. NaOH
 (+) result: yellow sol’n (heating); orange sol’n (x’ss NaOH)
 Principle: nitration of aromatic ring

- O H O -
O O
O
O

NH3 NH3 NH3

HNO 3 x' cess NaO OH -
OH O
N N
- -
O O
 Millon’s test
 Purpose: phenolic group in Tyrosine
 Reagents: HgSO4 in H2SO4
 (+) result: old rose/ flesh/ purple-red ppt.
 Principle: complexation (mercuration & nitration or nitrosation /
complexation of nitrohydroxyphenyl derivatives with Hg2+)
 Hopkins-cole test
 Purpose: indole group in Tryptophan (Trp)
 Reagents: glyoxylic acid & conc. H2SO4
 (+) result: pink to violet interface
Principle: reduction of oxalate to glyoxylate and acid-catalyzed
condensation of two Trps with glyoxylic acid
OH
OH
O
O
-
NH3 Mg O O

O O H 3N O O
-
O + OH +
H H H
N
O H
H N N
H
O

NH3
-
O
 Sakaguchi test
 Purpose: guanido group of Arginine
 Reagents: α-naphthol, NaOBr, NaOH & Urea
 (+) result: red to red-orange color
Principle: base-catalyzed condensation of α-naphthol with the
guanido group of arginine
O
H
H3N + -
O O
H
H3N + OH
-
O
- H O
OH N
NH + 2

N N N
NH 2

OH
 Fohl’s/Lead acetate test
 Purpose: S-containing amino acids ( M & C )
 Reagents: Lead (II) acetate & NaOH
 (+) result: brown or black ppt.
 Principle: degradation and substitution reaction to form PbS
 Pauly’s test/Diazo rxn
 Purpose: detects the presence of histidine and tyrosine
 Reagents: sulfanilic acid, NaNO2 & Na2CO3
 (+) result: red color
Principle: diazotized sulfanilic acid couple with amino phenol to
form a color red azo compound in cold condition
Pauly’s test/Diazo rxn
 Nitroprusside test
 Purpose: presence of –SH group (cysteine)
 Reagents: Na2Fe(CN)5NO in dilute NH3
 (+) result: red coloration
 Principle: complexation
 Amide test
 Purpose: It detects 1o, 2o, 3o amides and nitriles
 Reagents: NaOH
 (+) result: red litmus paper → blue
 Principle: Base-catalyzed Hydrolysis

• Recall:

NaOH
 Qualitative Color
Color Reactions
Reaction Intact Protein Hydrolysate
Protein
acidic basic enzymatic

Biuret +++ - - ++
Ninhydrin +/- +++ +++ ++
Xanthoproteic +++(F,Y,
+(Y) ++(F,Y) +++
W)
Millon’s +++ +++ +++ +++
Hopkin’s-Cole - - ++ ++
Sakaguchi +++ +++ - ++
Nitroprusside +/- +/- +/- ++
Fohl’s +/- +/- - +/-
Test for amide - - - ++
Pauly +++ +/- +++ +++
 Chromatography
• Used to separate and identify components/solutes in a
mixture
• Principle: based on affinity
• Planar Chromatography is classified as normal phase &
partition

• Mobile phase: ButOH:HOAc:H2O (4:1:5)

• Stationary phase: H2O molecules adsorb by cellulose
fibers
 Chromatography
• Used to determine the amino acid composition of a
given protein solution

• Visualized using ninhydrin

• Retention factor, RF
RF = distance travelled by the amino acid, cm
distance travelled by the solvent, cm
Chromatography
• Stages in Paper Chromatography
1. Sample/Std. Application
2. Development
3. Visualization
4. Evaluation
5. Documentation
Chromatography
 “Biochemical research often requires the
quantitative measurement of protein
concentrations in solutions. Several
techniques for such measurement have
been developed; however, most have
limitations because either they are not
sensitive enough or they are based on
reactions with specific amino acids in the
protein.” – (Boyer, 2012)

PROTEIN ASSAYS
 Bradford Assay
→ a simple colorimetric and one of the most commonly used
assay for total protein concentration
→ It is based on the proportional binding of Coomassie dye to
proteins
→ Principle involved: in an acidic medium, proteins bind
to coomassie dye due to hydrophobic and electrostatic
interactions.
→ λ max of dye = 465 to 595 nm
 Bradford Assay
→ involves use of Coomassie Brilliant Blue G-250 (dye), which
reacts primarily to basic (especially arginine) and aromatic
amino acids
→ measures 10-100 mg protein
→ standard used: bovine serum albumin (BSA)
→ Bradford reagent:
dye dissolved in ethanol and phosphoric acid
Bradford Assay
Bradford Assay
Steps in quantifying proteins using Bradford assay:
1. Prepare BSA standards with different concentration
- stock solution added with different amounts of water
- final concentration of standard is computed using
C1V1 = C2V2
2. Read A595 of standards and samples
3. Plot standard curve
concentration (y) vs. Absorbance (x)
4. Draw the best fit line
5. Determine the concentration of sample from the
standard curve by extrapolation

Gladys Ilagan Gen Biochemistry

Bradford Assay

http://www.bio-rad.com
Bradford Assay
Bradford Assay
Bradford Assay
Table 3. Types of Protein assays (summary)