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Isolation and

Characterization
of Proteins

Amino Acids

Nelson and Cox, 2004

Amino Acids

Nelson and Cox, 2004

Amino Acids

Nelson and Cox, 2004

Levels of Protein Structure

Nelson and Cox, 2004

Classification of Proteins Accdg. To


Composition
1. SIMPLE PROTEINS
- yield only amino acids upon hydrolysis
2. CONJUGATED PROTEINS
simple proteins + non-protein substances
ex. Nucleoprotein
Glycoprotein
Lipoprotein
Phosphoprotein
Hemoprotein
Metalloproteins

Accdg. To Biological
Function
1. CATALYST
2. TRANSPORT PROTEINS
3. NUTRIENT and STORAGE PROTEINS
4. STRUCTURAL PROTEINS
5. CONTRACTILE and MOTILE PROTEINS
6. DEFENSE PROTEINS
7. REGULATORY 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
- soluble in water and dilute aq. solutions
2. GLOBULINS
- soluble in dilute salt solutions but are insoluble or
sparingly soluble in water
3. GLUTELINS
- soluble in dilute solutions of acids and bases
- insoluble in neutral solvents
4. PROLAMINS
- soluble in 50-90% alcohol
- insoluble in water, neutral solvents or absolute
alcohol
5. ALBUMINOIDS/SCLEROPROTEINS
- 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. Temperature

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

INCOMPLETE HYDROLYSIS
-specific peptide bonds hydrolyzed by
proteases like:
Trypsin
Chymotrypsin
Pepsin
Bromelain
Papain

Separation/Purification of Proteins
Properties of proteins being considered:
1. Charge
2. Molecular size, shape
3. Solubility
4. Affinity to a ligand
5. pI

Casein
- Phosphoprotein (Phosphate groups
attached to OH groups of ser or thr) that
exists as calcium caseinate
- present as micelles in milk
- Serves as a storage protein in milk
- Isolated from milk by isoelectric
precipitation
- Isoelectric pH 4.6

Isolation of Casein and Albumin from Cows Milk


Cows milk
Milk proteins

Casein
alpha-s1
alpha-s2
beta
kappa

Whey proteins
alpha-lactalbumin
beta-lactoglobulin
serum albumin
immunoglobulins
other proteins

Alpha-Lactalbumin
- 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
- isolated from whey by heat denaturation
(in acidic condition)

Gladys Ilagan Gen Biochemistry

Gladys Ilagan Gen Biochemistry

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
- Isolated by salt precipitation

Myoglobin

Gladys Ilagan Gen Biochemistry

Gladys Ilagan Gen Biochemistry

Gluten
- Storage protein responsible for the
elasticity and extensibility of dough
- consists of gliadin and glutenin
- Isolated by difference in solubility in
water
- Isolated gluten free of starch when
(-) to iodine test

Qualitative Color Reactions


1. BIURET TEST
-test for presence of a peptide bond
(peptide must have at least 3 amino acids)

Reagent:
Principle:

CuSO4 + NaOH

Principle: formation of
coordination complex of Cu2+ and
four nitrogen atoms (two from
each of the two polypeptide
chains)
(+) Result: purple color of solution

Qualitative Color Reactions


2. NINHYDRIN TEST
-test for a-amino acid
Reagent:
Principle:

(+) Result:

Ninhydrin (triketohydrindene
hydrate)
oxidative decarboxylation &
deamination followed by
condensation
blue-violet color
yellow for proline
(pyrrolidine ring)

Qualitative Color Reactions


3. XANTHOPROTEIC TEST
-test for aromatic amino acids
Reagent:

HNO3 , NaOH

Principle:

nitration of aromatic rings


via SEAr

(+) Result:

yellow color of solution


with HNO3
orange color of solution
with NaOH

Qualitative Color Reactions


4. MILLONS TEST
-test for tyrosine

Reagent:
Principle:

(+) Result:

salt of Hg dissolved in HNO3


complexation (mercuration &
nitration or nitrosation/
complexation of
nitrohydroxyphenyl derivatives
with Hg2+)
red color

Qualitative Color Reactions


5. HOPKINS-COLE TEST
-test for tryptophan
Reagent:
Principle:

(+) Result:

glacial CH3COOH, glyoxylic


acid, concd H2SO4
reduction of oxalic acid to
glyoxylic acid and acidcatalyzed condensation of two
tryptophans with glyoxylic acid
purple color at the interface

Qualitative Color Reactions


6. SAKAGUCHI TEST
-test for arginine
Reagent:

a-naphthol, NaOBr, NaOH, urea


(to stabilize color & destroy
excess OBr- ions)

Principle:

complexation (base-catalyzed
condensation of a-naphthol
with the guanido group of Arg)

(+) Result: red color

Qualitative Color Reactions


7. NITROPRUSSIDE TEST
-test for S-containing amino acids

Reagent:

NaOH, nitroprusside

Principle: Complexation reaction


(+) Result: red color

Qualitative Color Reactions


8. FOHLS TEST
-test for S-containing amino acids

Reagent:

NaOH, (CH3COO)2Pb

Principle:

degradation & substitution


reaction to form PbS
dark brown / black
precipitate

(+) Result:

Qualitative Color Reactions


9. TEST FOR AMIDE
- asn, gln
Reagent:

NaOH

Principle:

Basic hydrolysis

(+) Result:

evolution of gas, presence


tested using a litmus paper

Qualitative Color Reactions


10. PAULY TEST
-test for histidine and tyrosine
Reagent:

sulfanilic acid in NaNO2


solution,Na2CO3

Principle:

Formation of azo dyes

(+) Result:

Red color

Qualitative Color Reactions


Color Reaction

Biuret
Ninhydrin
Xanthoproteic
Millons
Hopkins-Cole
Sakaguchi
Nitroprusside

Fohls
Test for amide
Pauly

Intact
Protein

Protein Hydrolysate
acidic

basic

enzymatic

Paper 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

Stages in Paper Chromatography


Sample/Standard Application: small spots of
std/sample are applied to avoid overlapping and
tailing during development
Development: equilibration (saturation of
chamber with mobile phase) to hasten
development
Visualization: chemical visualizing agent:
ninhydrin spray for amino acids and proteins
Evaluation: comparing Rf values of sample and
standards
Documentation: chromatogram

Bradford Assay
-colorimetric method for determining protein
concentration
- 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
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
Absorbance (y) vs. Concentration (x)
4. Draw the best fit line
5. Determine the concentration of sample from the
standard curve by extrapolation

Bradford Assay

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