Download - Isolation and Characterization of Proteins
-
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 Whey proteins
alpha-s1 alpha-lactalbumin alpha-s2 beta-lactoglobulin beta serum albumin kappa 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: CuSO4 + NaOH
Principle: 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: Ninhydrin (triketohydrindene hydrate)
Principle: oxidative decarboxylation & deamination followed by condensation
(+) Result: 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: salt of Hg dissolved in HNO3
Principle: complexation (mercuration & nitration or nitrosation/ complexation of nitrohydroxyphenyl derivatives with Hg2+)
(+) Result: red color
-
Qualitative Color Reactions
5. HOPKINS-COLE TEST
-test for tryptophan
Reagent: glacial CH3COOH, glyoxylic acid, concd H2SO4
Principle: reduction of oxalic acid to glyoxylic acid and acid-catalyzed condensation of two tryptophans with glyoxylic acid
(+) Result: 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
(+) Result: dark brown / black precipitate
-
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 Intact Protein
Protein Hydrolysate
acidic basic enzymatic
Biuret
Ninhydrin
Xanthoproteic
Millons
Hopkins-Cole
Sakaguchi
Nitroprusside
Fohls
Test for amide
Pauly
-
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
http://www.bio-rad.com