Isolation and Characterization of Proteins

Isolation and Characterization

of Proteins

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

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

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


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)


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


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


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


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)



7. NITROPRUSSIDE TEST

-test for S-containing amino acids

Reagent: NaOH, nitroprusside

Principle: Complexation reaction



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


9. TEST FOR AMIDE

- asn, gln

Reagent: NaOH

Principle: Basic hydrolysis

(+) Result: evolution of gas, presence tested using a litmus paper


10. PAULY TEST

-test for histidine and tyrosine

Reagent: sulfanilic acid in NaNO2 solution,Na2CO3

Principle: Formation of azo dyes

(+) Result: Red color


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

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Isolation and Characterization of Proteins