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Page 1: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

Lab Activity 1

Qualitative Tests for Amino Acids

IUG, 2013Dr. Tarek M. Zaida

Page 2: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

Main Classes of food ?

• Carbohydrates: (readily available energy resources)

• Lipids: (principal energy reserves)

• Proteins: (Source of energy for growth & cellular maintenace)

Page 3: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

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.

Page 4: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

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).

Page 5: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

Classification of amino acids

• Non-essential..Are synthesized by the body

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

Page 6: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

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

Page 8: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

Experiments

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

Seven Tests:1. Ninhydrin 4. Xanthoproteic 7. Sakaguchi2. Biuret 5. Hopkin’s- Cole3. Millon’s 6. Sulfur

Page 9: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

1. Ninhydrin Test

• For amino acids containing a free NH2 & free COOH.

• Reaction with ninhydrin to produce a colored product.

1. When NH2 is attached to α-C on the amino acid’s carbon chain, the amino group’s N is part of a blue-purple product.

2. Amino acids that have N-H (a secondary amino group (e.g. proline) also react with ninhydrin, but they yield a yellow product.

Page 10: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

Reaction of A.A with Ninhydrin

Page 11: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

Procedure..

1. Label 6 cleaned, drained test tubes with the names of the following solutions: 2 % glycine, 1 % tyrosine, 2 % proline, 2 % casein, 2 % gelatin, 2 % albumin.

2. Add 15 drops of each solution in the corresponding test tube.3. To each of the test tubes add 5 drops of 0.5 % ninhydrin reagent solution.

4. Place the test tubes into the boiling-water bath for 5 minutes. Remove the test tubes from the water bath and place then in a test

tube rack. Record your observations!

Page 12: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

2. Biuret

• For detecting peptide bonds (hence peptides or proteins)..

• How it works?• The copper atoms of Biuret solution (CuSO4 ) in a

basic environment will react with peptide bonds (-CO ---NH) to form a chelate of a deep violet color, indicating the presence of proteins.

• A light pink color indicates the presence of peptides..

Page 13: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

Biuret complex with proteins…

Page 14: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

Procedure..

1. To 1 ml of a solution containing protein add 4 ml of a biuret reagent.2. Mix well, then let to stand at RT for about 30 min.3. Record your abservations!

Page 15: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

3. 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.

Page 16: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

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.

Page 17: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

4. 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.

Page 18: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

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

Page 19: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

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.

Page 20: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

5. 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

Page 21: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

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.

Page 22: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

6. Sulfur Test

• For the detection of sulfur-containing amino acids such as cysteine.

• Is done by converting S to an inorganic sulfide ( S2-) through cleavage by a base.

• When the resulting solution is combined with lead acetate (CH3COOPb), a black precipitate of lead sulfide is formed.

Sulfur-containing protein ----> NaOH----> S2- ----Pb2+----> PbS

Cysteine

Page 23: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

Procedure..

1. Place 1 ml of 2% casein, 2% egg albumin, 2% peptone, 2% gelatine and 0.1 M cysteine into separate, labeled test tubes.

2. Add 2 ml of 10 % aqueous sodium hydroxide. Add 5 drops of 10 % lead acetate solution.

3. Stopper the tubes and shake them. Remove the stoppers and heat in a boiling water bath for 5 minutes. Cool and record the results.

Page 24: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

7. Sakaguchi

• For detection of the amino acid containing the guanidinium group (e.g. arginine).

• In basic conditions, α- naphthol and sodium hypobromite/chlorite react with the guanidinium group to form red orange complexes.

Guanidinium group

Arginine

Page 25: Lab Activity 1 Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida Qualitative Tests for Amino Acids IUG, 2013 Dr. Tarek M. Zaida.

Procedure

1. Add 1 ml of 3 N NaOH solution to 1 ml of the protein solution, followed by addition of 0.5 ml of 0.1 % α- naphthol solution, and a few drops of 2 % hypobromite solution (NaOBr).

2. The formation of a red color indicates the presence of a guanidinium group in the compound under examination.