By: Dr. Beenish Zaki Senior Instructor Department of Biochemistry. 04/10/12

 As you get your results start writing your observation in the observation column. The inference and Principle will be discussed at the end of the session. XanthoproteicTest PrincipleObservationInference

 Take 3 ml of Protein Solution in a test tube.  Add 3ml of 5% Na OH  Add a drop of 1 % CuSO 4  Mix Well.

 It is due to coordination of cupric ions with peptide nitrogen and O 2 of water to form a coloured co-ordination complex in an alkaline medium.  A minimum two peptide bonds should be present for the Biuret test to be positive.

Biuret (-)ve test (+) test (+) test Reagent blue colour violet colour pink colour

 Take 1 ml of Protein Solution and add 1ml of Conc. HNO 3  Boil for 2 minutes.  Cool it under tap water.  Then add 3ml of 40%NaOH. Mix Well

 Test for Aromatic Amino Acids.  Nitration of Aromatic ring occurs on boiling with HNO 3. This in Alkaline medium forms a coloured complex.  Examples: Tyrosine, Phenylalanine.

Tyrosine

 Take 1ml of protein solution.  Add 1ml of Millon’s reagent mix and boil for 1 minute.  Cool it.  Add 3 drops of 1%NaN0 2.  Mix Well.

 MILLON’S REAGENT IS COMPOSED OF: ◦ 10% Hg SO 4 in 10 % H 2 SO 4

 This is a test for Tyrosine  This test is carried out in a strongly acidic medium to prevent precipitation of mercury.  Sodium nitrite reacts with sulphuric acid to form nitrous acid. The reaction involves the mercuration of Tyrosine and on boiling it produces a coloured complex.

 These test are used for screening of inborn errors of metabolism.(Aminoacidurias)

 Each student needs: ◦ Gloves ◦ Test Tube rack ◦ Test tube holder ◦ Test tube (3 in number)

PrincipleObservationInference Violet colour is obtained Protein is present in the solution.

PrincipleObservationInference Yellow colour observed Aromatic amino acid present

PrincipleObservationInference pink colour is obtained Tyrosine is present