Colour Reaction of Amino Acids

Prepared by:Group#2

Quinto, JasmineSta. Isabel, Rizzalyn

Yadao , MoniqueNiazmand Saravani, MostafaNiazmand Saravani, Mojtaba

Seong, Jo-eunMajidzadeh, 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 molecule2.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 testThis 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 testAmino 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 testThis 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 testThe 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 testThe 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 alpha-naphthanol 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.

Objectives1.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

Apparatus1. Hot plate/ stove2. Alcohol lamp3. 600mL beaker4. 50mL beaker5. Test tubes6. Test tube rack

1. 10mL graduated cylinder

2. Pipets3. Droppers4. Litmus paper5. 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

ProteinsThe 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 test1. 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%NaOH3. Add 2 drops of CuSO4 solution.

Observe the result.4. Run a blank test with distilled

water.

Ninhydrin test1. 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%ninhydrin3. Let cool and observe

4. Run a blank test with distilled water.

Xanthoproteic test1. 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 change5. 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 test1. 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 Millon’s 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 test1. 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 Reaction1. 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 SolutionsColour Test Saliva

solutionEgg Albumin Gly Tyr Trp Arg CYs

Biuet test XXXX XXXX XXXX XXXX

Ninhydrin XXXX XXXX XXXX XXXX

Xantho-proteic

XXXX XXXX

Millon test XXXX XXXX XXXX

Bromine water

XXXX XXXX XXXX

Unoxidized XXXX XXXX XXXX

Sakaguchi XXXX XXXX XXXX

Hopkins-cole

XXXX XXXX XXXX