1. Lab activity 8 Proteins 2 Alaa S Baraka Islamic university of Gaza March2013

2. Color test of amino acids and proteins A.A can be characterized qualitatively by using several dyes that will react with certain groups of the A.A.

3. Lab activity Sulfer test Sakaguchi test Ninhydrin test Biuret test

4. Sulfur test (Test for (-SH) group) (To detect amino acid which containe sulfur group)

5. Principle Proteins containing sulfur (cysteine and cystine and Methionine) give a black deposit of lead sulfide (PbS) when heated with lead acetate in alkaline medium. Sulfur-containing protein ----> NaOH----> S2- ----Pb2+----> PbS

6. Procedure 1.Add 1 ml of protein solution in a test tube, add 2 ml of 10% sodium hydroxide solution and 5 drops of lead acetate. 2.labble the tubes and shake them. then heat in a boiling water bath for 5 minutes. 3.Cool and record the results.

7. Result: A black deposit is formed with albumin while a slight black turbidity is obtained with casein due to its lower content of sulfur. Gelatin gives negative result.

8. Sakaguchi Test for Arginine ( detection of a specific type of protein with the amino acid containing the guanidinium group ) guanidinium group

9. Principle: α- naphthol and sodium hypobromite/chlorite react with guanidine group to form red orange complexes. NaOBr+ >>>>>>>>>>>> َ Quinon Quinon+ guanidine >>>>>>>>>>> Red complex

10. Procedure 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).

11. Biuret ( For detecting peptide bonds )

12. Principle The copper atoms of Biuret solution (CuSO 4 ) 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..

13. Biuret complex with proteins

14. 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.

15. Ninhydrin Test ( general test for compounds with free a – amino groups) For amino acids containing a free NH 2 & free COOH. Reaction with ninhydrin to produce a colored product. 1.When NH 2 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.

16. Reaction of A.A with Ninhydrin

17. Procedure..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! 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! 17