1. Lab Activity 8 Proteins part II IUG, Spring 2014 Dr. Tarek Zaida 1

2. Experiments A.A can be characterized qualitatively by using several dyes that will react with certain groups of the A.A.  Seven Tests: 1.Ninhydrin 4. Xanthoproteic 7. Sakaguchi 2.Biuret5. Hopkin’s- Cole 3.Millon’s6. Sulfur A.A can be characterized qualitatively by using several dyes that will react with certain groups of the A.A.  Seven Tests: 1.Ninhydrin 4. Xanthoproteic 7. Sakaguchi 2.Biuret5. Hopkin’s- Cole 3.Millon’s6. Sulfur 2

3. 1. Ninhydrin Test 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. 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. 3

4. Reaction of A.A with Ninhydrin 4

5. 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! 5

6. 2. Biuret For detecting peptide bonds (hence peptides or proteins).. How it works? 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.. For detecting peptide bonds (hence peptides or proteins).. How it works? 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.. 6

7. Biuret complex with proteins… 7

8. 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. 3. Record your observations! 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. 3. Record your observations! 8

9. 3. Sulfur Test For the detection of sulfur-containing amino acids such as cysteine. Is done by converting S to an inorganic sulfide ( S 2- ) through cleavage by a base. When the resulting solution is combined with lead acetate (CH 3 COOPb), a black precipitate of lead sulfide is formed. Sulfur-containing protein ----> NaOH----> S 2- ---- Pb 2+ ----> PbS For the detection of sulfur-containing amino acids such as cysteine. Is done by converting S to an inorganic sulfide ( S 2- ) through cleavage by a base. When the resulting solution is combined with lead acetate (CH 3 COOPb), a black precipitate of lead sulfide is formed. Sulfur-containing protein ----> NaOH----> S 2- ---- Pb 2+ ----> PbS Cysteine 9

10. Procedure.. 1. Place 1 ml of 2% casein, 2% egg albumin, 2% peptone, 2% gelatine and 0.1 M cysteine into separate, labeled test tubes. 2. Add 2 ml of 10 % aqueous sodium hydroxide. Add 5 drops of 10 % lead acetate solution. 3. Stopper the tubes and shake them. Remove the stoppers and heat in a boiling water bath for 5 minutes. Cool and record the results. 1. Place 1 ml of 2% casein, 2% egg albumin, 2% peptone, 2% gelatine and 0.1 M cysteine into separate, labeled test tubes. 2. Add 2 ml of 10 % aqueous sodium hydroxide. Add 5 drops of 10 % lead acetate solution. 3. Stopper the tubes and shake them. Remove the stoppers and heat in a boiling water bath for 5 minutes. Cool and record the results. 10

11. 7. Sakaguchi For detection of the amino acid containing the guanidinium group (e.g. arginine). In basic conditions, α- naphthol and sodium hypobromite/chlorite react with the guanidinium group to form red orange complexes. For detection of the amino acid containing the guanidinium group (e.g. arginine). In basic conditions, α- naphthol and sodium hypobromite/chlorite react with the guanidinium group to form red orange complexes. Guanidinium group Guanidinium group Arginine 11

12. Procedure 1.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). 2. The formation of a red color indicates the presence of a guanidinium group in the compound under examination. 1.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). 2. The formation of a red color indicates the presence of a guanidinium group in the compound under examination. 12