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Lab Activity 7Proteins

Part I

IUG, Spring 2014Dr. Tarek Zaida

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1. Isolation of Casein from Cow milk

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Background

Milk composition

Carbohydrates

Lipids

ProteinsVitamins

Minerals

Water

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Casein

α β κ

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Casein MW

Kd

Phosphate

groups/molecule

α 27.3 9

β 24.1 4-5

κ 8.0 2

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Casein can be precipitated by:1. Calcium ions2. HCl 3. Renin4. Bacteria

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Experiment1: Isolation of Casein from cow milk

• Reagents• 20 ml milk, • Glacial acetic acid (100%), • Ethanol (95% v/v), • Ether, Thermometer to 100 oc.

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Procedure

1. Place 20 ml (20 g) of milk into a 125 ml flask and heat at 40 oC in a water bath.

2. Add 5 drops of glacial acetic acid and stir for about 1 min.

3. Filter the resulting mixture through 4 layers of cheesecloth held in a funnel and gently squeeze out most of liquid.

4. Remove the solid (casein and fat) from the cheesecloth, place it into a 100 ml beaker and add 10 ml of 95% ethanol.

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5. Stir well to break up the product. Pour off the liquid and add 10 ml of 1:1 ether-ethanol mixture to the solid. Stir well and filter through 4 layers of cheesecloth.

6. Let the solid drain well, then scrape it into a weighed filter paper and let it dry in the air.

• Calculate the casein percentage in milk as follows: % Casein =__grams of casein__ x 100

• grams of milk

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Color Reactions of Proteins

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Proteins are….

• The most important cell content after water• Are either functional or structural • Macromolecules made up of amino acids,

connected together by peptide bonds.

Peptide bond: Amide bond, formed between COOH & -NH2 of 2 adjacent amino acids.

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Amino acids

Proteins are made up of 20 A.A. All of them have the same general structural formula shown above, however they are different in the R- group (side chain).

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Classification of amino acids

• Non-essential..Are synthesized by the body

• Essential..(Valine, Leucine, Isoleucine, Methionine, Threonine, Tryptophan, Phenylalanine, Lysine, Histidine)

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What amino acids chemical reactions are due to?

1. Amphoteric nature2. R-group or side chain

The accessibility of certain functional groups to the reagent will determine the intensity of the product color.

The color intensity varies among proteins and is proportional to the number of reacting functional, or free groups present.

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A.A in acidic, neutral, and basic solutions

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Experiments

• A.A can be characterized qualitatively by using several dyes that will react with certain groups of the A.A.

Tests:1. Millon’s 2. Xanthoproteic 3. Hopkin’s- Cole (glyoxylic Acid)

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1. Millon’s

• Any compound containing a phenolic hydroxyl group will give a positive result with Millon’s reagent.

• Cosequently..• Proteins containing tyrosine will give a positive

test of a pink to dark-red color• Note: Some proteins will initially form a white

precipitate that will turn red when heated.

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Procedure..

1. 2 ml of 2% casein, 2% egg albumin, and 0.1 M tyrosine add 3 drops of Millon's reagent.2. Immerse the tubes in a boiling water bath for 5 minutes. 3. Cool the tubes down. Record the colors formed.

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2. Xanthoproteic

For detection of aromatic groups, derivative of benzene, (hence aromaric amino acids).

These aromatic groups can undergo reactions characteristic of benzene, and its derivatives.

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One such a characteristic reaction for benzene is: Nitration

The amino acids tyrosine and tryptophan contain activated benzene rings and readily undergo nitration, while phenylalanine does not contain a readily activated benzene ring.

a. Act. tyrosine b. Act. Tryptophan

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Procedure...

1. Add 1 ml of a concentrated HNO3 in a test tube containing 2 ml of a protein solution.2. The formed white precipitate, will turn yellow upon heating, and finally will dissolve giving a yellow color to the solution.3. Cool the solution down. Carefully add 3 ml of 6 N NaOH. The yellow color turns orange.

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3. Hopkins-Cole (Glyoxylic Acid Reaction)

• Specific for tryptophan (the only amino acid containing indole group)

• Reacting with a glyoxylic acid in the presence of a strong acid, the indole ring forms a violet cyclic product.

• The protein solution is hydrolyzed by conc. H2SO4 at the solution interface.

• Once the tryptophan is free, it reacts with glyoxylic acid to form violet product.

Indole

Glyoxylic acid

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Procedure..

1. In a test tube, add to 2 ml of the solution under examination, an equal volume of Hopkins- Cole reagent and mix thoroughly.

• Incline the tube and let 5 to 6 ml of conc. H2S04 acid flow slowly down the side of the test tube, thus forming a reddish - violet ring at the interface of the two layers. That indicates the presence of tryptophan.