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Lab Activity 8Proteins part II

IUG, Spring 2014Dr. Tarek Zaida

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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. Sakaguchi2. Biuret 5. Hopkin’s- Cole3. Millon’s 6. Sulfur

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1. Ninhydrin Test

• For amino acids containing a free NH2 & free COOH.

• Reaction with ninhydrin to produce a colored product.

1. 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.

2. Amino acids that have N-H (a secondary amino group (e.g. proline) also react with ninhydrin, but they yield a yellow product.

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Reaction of A.A with Ninhydrin

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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!

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2. Biuret

• For detecting peptide bonds (hence peptides or proteins)..

• How it works?• 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..

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Biuret complex with proteins…

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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!

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3. Sulfur Test

• For the detection of sulfur-containing amino acids such as cysteine.

• Is done by converting S to an inorganic sulfide ( S2-) through cleavage by a base.

• When the resulting solution is combined with lead acetate (CH3COOPb), a black precipitate of lead sulfide is formed.

Sulfur-containing protein ----> NaOH----> S2- ----Pb2+----> PbS

Cysteine

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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.

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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.

Guanidinium group

Arginine

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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.