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Lab Activity 7 Proteins Part I

Lab Activity 7 Proteins Part I. IUG, Spring 2014 Dr. Tarek Zaida. Background. Casein can be precipitated by: 1. Calcium ions 2. HCl 3. Renin 4. Bacteria. Experiment1: Isolation of Casein from cow milk. Reagents 20 ml milk, Glacial acetic acid (100%), Ethanol (95% v/v),

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Lab Activity 7 Proteins Part I

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  1. Lab Activity 7Proteins Part I IUG, Spring 2014 Dr. TarekZaida

  2. Background

  3. Casein can be precipitated by: 1. Calcium ions 2. HCl 3. Renin 4. Bacteria

  4. Experiment1: Isolation of Casein from cow milk • Reagents • 20 ml milk, • Glacial acetic acid (100%), • Ethanol (95% v/v), • Ether, Thermometer to 100 oc.

  5. 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.

  6. 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

  7. Color Reactions of Proteins

  8. 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.

  9. 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).

  10. Classification of amino acids • Non-essential.. Are synthesized by the body • Essential.. (Valine, Leucine, Isoleucine, Methionine, Threonine, Tryptophan, Phenylalanine, Lysine, Histidine)

  11. What amino acids chemical reactions are due to? 1. Amphoteric nature 2. 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.

  12. A.A in acidic, neutral, and basic solutions

  13. Experiments • A.A can be characterized qualitatively by using several dyes that will react with certain groups of the A.A. • Tests: • Millon’s • Xanthoproteic • Hopkin’s- Cole (glyoxylic Acid)

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. Procedure.. • 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.

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