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Lab Activity 8 Proteins part II. IUG, Spring 2014 Dr. Tarek Zaida. Experiments. A.A can be characterized qualitatively by using several dyes that will react with certain groups of the A.A. Seven Tests: Ninhydrin 4. Xanthoproteic 7. Sakaguchi Biuret 5. Hopkin’s - Cole

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lab activity 8 proteins part ii

Lab Activity 8Proteins part II

IUG, Spring 2014

Dr. TarekZaida

experiments
Experiments
  • A.A can be characterized qualitatively by using several dyes that will react with certain groups of the A.A.
  • Seven Tests:
  • Ninhydrin 4. Xanthoproteic 7. Sakaguchi
  • Biuret 5. Hopkin’s- Cole
  • Millon’s 6. Sulfur
1 ninhydrin test
1. Ninhydrin Test
  • 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.
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.

  • 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!

2 biuret
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..
procedure1
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!

3 sulfur test
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

procedure2
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.

7 sakaguchi
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

procedure3
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).

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

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