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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.