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Lab activity 8 Proteins 2

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  1. Lab activity 8Proteins 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 • Add 1 ml of protein solution in a test tube, add 2 ml of 10% sodium hydroxide solution and 5 drops of lead acetate. • labble the tubes and shake them. then heat in a boiling water bath for 5 minutes. • 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 (CuSO4 ) 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 NH2 & free COOH. • Reaction with ninhydrin to produce a colored product. • When NH2 is attached to α-C on the amino acid’s carbon chain, the amino group’s N is part of a blue-purple product. • 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.. 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. • To each of the test tubes add 5 drops of 0.5 % ninhydrin reagent solution. • 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

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