1. Lab Activity 7 Proteins Part I IUG, 2015 Dr. Tarek Zaida 1

2. 1. Isolation of Casein from Cow milk 2

3. Background Milk composition CarbohydratesLipidsProteinsVitaminsMineralsWater 3

4. 4

5. Casein MW Kd Phosphate groups/molecule α27.39 β24.14-5 κ8.02 5

6. Casein can be precipitated by: 1. Calcium ions 2. HCl 3. Renin 4. Bacteria 6

7. Experiment1: Isolation of Casein from cow milk Reagents 20 ml milk, Glacial acetic acid (100%), Ethanol (95% v/v), Ether, Thermometer to 100 o c. Reagents 20 ml milk, Glacial acetic acid (100%), Ethanol (95% v/v), Ether, Thermometer to 100 o c. 7

8. Procedure 1. Place 20 ml (20 g) of milk into a 125 ml flask and heat at 40 o C 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. 1. Place 20 ml (20 g) of milk into a 125 ml flask and heat at 40 o C 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. 8

9. 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 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 9

10. Color Reactions of Proteins 10

11. 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 & -NH 2 of 2 adjacent amino acids. 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 & -NH 2 of 2 adjacent amino acids. 11

12. 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). 12

13. Classification of amino acids Non-essential.. Are synthesized by the body Essential.. (Valine, Leucine, Isoleucine, Methionine, Threonine, Tryptophan, Phenylalanine, Lysine, Histidine) Non-essential.. Are synthesized by the body Essential.. (Valine, Leucine, Isoleucine, Methionine, Threonine, Tryptophan, Phenylalanine, Lysine, Histidine) 13

14. 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. 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. 14

15. A.A in acidic, neutral, and basic solutions 15

16. 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) 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) 16

17. 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. 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. 17

18. 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. 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. 18

19. 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.  For detection of aromatic groups, derivative of benzene, (hence aromaric amino acids).  These aromatic groups can undergo reactions characteristic of benzene, and its derivatives. 19

20. 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  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 20 phenylalanine

21. Procedure... 1. Add 1 ml of a concentrated HNO 3 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. 1. Add 1 ml of a concentrated HNO 3 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. 21

22. 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. H 2 SO 4 at the solution interface. Once the tryptophan is free, it reacts with glyoxylic acid to form violet product. 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. H 2 SO 4 at the solution interface. Once the tryptophan is free, it reacts with glyoxylic acid to form violet product. Indole Glyoxylic acid 22

23. 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. H 2 S0 4 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. 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. H 2 S0 4 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. 23