Colour Reaction of Amino Acids

Prepared by: Group#2 Quinto, Jasmine Sta. Isabel, Rizzalyn Yadao , Monique Niazmand Saravani, Mostafa Niazmand Saravani, Mojtaba Seong, Jo-eun Majidzadeh, Hossein

Proteins are complex, high molecular weight biomolecules composed of amino acids joined together by peptide bonds. The twenty amino acids normally found in proteins differ in their amino groups in the side chains. These respond to specific qualitative tests with certain chemical compounds chemical reagents and thus become the basis of their detection whether as free amino acids or in combined form as in peptides or proteins

Reactions of proteins may be divided into three categories: 1. Reaction due to the presence of specific chemical groups are linkages in the protein molecule 2. Reaction due to the acidity or basicity of the protein 3. Reactions due to the colloidal nature of the proteins

Colour Reactions of Amino Acids and Proteins

Biuret test
This is the test indicates the presence of peptide linkages. The purplish to violet colour is apparently due to the cupric ions with the unshared electron pairs of four nitrogen atoms. All substance is proportional to the number of peptide bonds give the test and the intensity of the purple colour produced id proportional to the number of peptide bonds present.

Ninhydrin test
Amino acids reacts with ninhydrin (tryketoninhydrindene hydrate) to yield CO2, NH3 and aldehyde containing one less carbon than the amino acid. The reaction is also yields a blue or purple colour useful for the colorimetric determination of amino acids.

Xanthoproteic test
This test is positive for proteins and amino acids containing an aromatic side chain (phenylalanine, tyrosine, and tryptophan). The benzene ring undergoes nitration with concentrated HNO3 giving nitro derivatives which are yellow in colour. Phenylalanine does not response readily to this test and requires H2SO4 as catalyst.

Millon-Nasse test
The phenolic group of tyrosine reacts with Millon-Nasse reagent (HgSO4 in H2SO4) forming an old rose or pink to red complex upon heating. The complex is probably the mercury salt of the phenolic compound.

Bromine water test

The pyrrole ring of tryptophan undergoes halogenation with Br2 water in the presence of amyl alcohol giving a pink or lavender colour in the alcohol layer.

Unoxidized sulfur test

This is given by the sulfur containing amino acids cysteine and cystine (methionine is quite stable alkaline lead acetate) whn boiled in strongly alkaline solutions. The liberated sulfur reacts with PbAc2 forming a brown to black precipitate of lead sulfide.

Sakaguchi Reaction
The guanidinium group of arginine in alkaline solution gives an orange or red colour with alphanaphthanol and sodium hypobromite

Hopkins-Cole test
The indole ring of tryptophan reacts with Hopkins-Cole reagent (glyoxylic acid) and sulfuric acid to produce a violet or ed purple color.

Objectives
1. To carry out the different colour reaction testes for amino acids and proteins; 2. Ro characterize amino acids based on their reactions with specific chemical reagents; 3. Ro identify the functional group of the amino acid responsible for the positive colour reaction.

Materials
 1%solution of egg albumin  0.1% solution of tryptophan  Arginine  Cysteine  Tryrosine  Phenylalanine  10% formic acid solution  0.1% ninhydrin  5% CuSO4  Saturated lead acetate  10% NaOH  Millon-Nasse reagent  Bromine water  N-amyl alcohol  Alpha-naphtanol solution  20%NaOH  Hopkins-Cole reagent

Apparatus
1. 2. 3. 4. 5. 6. Hot plate/ stove Alcohol lamp 600mL beaker 50mL beaker Test tubes Test tube rack 1. 2. 3. 4. 5. 10mL graduated cylinder Pipets Droppers Litmus paper Wire guaze

Procedure

Preparation of Saliva Solution

1. Collect 10mL of saliva using a clean 50mL beaker 2. Dilute the saliva with an equal volume of distilled water

Colour Reaction of Amino Acids and Proteins

The following tests will be performed on acid soluble proteins of saliva solution and 1% solution of egg albumin. Some or the tests may require to be performed on specific amino acids. Ain all of the following tests, perform a blank test with distilled water instead of the protein sample.

Biuret test
1. In 2 different test tubes, place1mL each of the protein samples (saliva solution and 1% solution of egg albumin) 2. To each test tube add 1mL of 10%NaOH 3. Add 2 drops of CuSO4 solution. Observe the result. 4. Run a blank test with distilled water.

Ninhydrin test
1. In 2 different test tubes, place1mL each of the protein samples (saliva solution and 1% solution of egg albumin) 2. Heat each of the protein solutions with 0.25mL of 0.1%ninhydrin 3. Let cool and observe 4. Run a blank test with distilled water.

Xanthoproteic test
1. In 2 different test tubes, place1mL each of the protein samples (saliva solution and 1% solution of egg albumin) 2. To each protein solution, add 1mL of concentrated HNO3. 3. Note if a heavy white precipitate is formed. Heat carefully to boiling and observe the colour of the solution/precipitate. 4. Cool and make alkaline with 10% NaOH. Note the colour change 5. Cool and make alkaline with 1% solutions of tryptophan, tyrosine, and phenylalanine in different test tubes. 6. Run a blank test with distilled water.

Millon-Nasse test
1. In 2 different test tubes, place1mL each of the protein samples (saliva solution and 1% solution of egg albumin) 2. Add 2 to 3 drops of Millons reagent to each of the protein solutions. 3. Boil in a boiling water bath for 5 minutes. Observe. 4. Perform an identical test with 0.1% tryptophan solution. 5. Run a blank test with distilled water.

Bromine water test

1. In 2 different test tubes, place1mL each of the protein samples (saliva solution and 1% solution of egg albumin). And add 3 drops of bromine water and 1mL of n-amyl alcohol. Shake well 2. Observe the colour of the alcohol layer. 3. Repeat the steps in with the 1% tryptophan solution 4. Run a blank test with distilled water.

Unoxidized sulfur test

1. Mix 1mL each protein samples in 3 different tubes with 1mL each of 10% NaOH and saturated lead acetate, PbAc2. 2. Boil in a water bath for 3 minutes and observe the result. 3. Perform an identical test with 0.1% cystein and a bank test with distilled water.

Sakaguchi Reaction
1. Place 1mL each of protein samples in 3 different test tubes and add 2mL each of 20% NaOH and alpha naphtanol solution. 2. Mix thoroughly and add 0.5mL of freshly prepared bromine water. Observe the result. 3. Perform an identical test with 0.1% cystein and a bank test with distilled water.

Hopkins-Cole test
1. Place 2mL each of sample solution in 3 different test tubes and to each tube add 1mL of Hopkins-Cole reagent. 2. Incline the tube and carefully add along the side 2mL at concentrated H2SO4. 3. Observe the colour at the junction of two liquids. 4. Perform an identical test with 0.1% trytophan.

Test Solutions
Colour Test Saliva solution
Egg Albumin

Gly

Tyr

Trp

Arg

CYs

Biuet test Ninhydrin Xanthoproteic

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Millon test Bromine water Unoxidized Sakaguchi Hopkinscole

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