Documente Academic
Documente Profesional
Documente Cultură
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
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.
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
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.
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
XXXX XXXX
XXXX XXXX
XXXX XXXX