Tests for Proteins -2

1. Xanthoproteic Test

Solution contents:
Conc. HNO₃

Tested for:
activated benzene rings. ( Tyrosine and Trptophan)

Theory and observations:
The aromatic groups in the amino acids can undergo nitration with nitric acid and give in yellow coloured products as shown below if the benzene ring is activated. Phenylalanine, though a derivative of benzene doesn’t undergo nitration because it’s not activated like tryptophan and tyrosine.

If you accidentally spill any nitric you may experience yellow stains because of this xanthoproteic reaction because the epidermal cells of the skin may contain proteins with aromatic amino acids.

2. Sakaguchi Test

Solution contents:
10% NaOH and Cl₂ /Br₂ [Sodium hypochlorite/bromide], α- napthol

Tested for:
Guanidium group. ( Arginine)

Theory and observations:
Arginine, the only amino acid with a guanidium group gives a red colour with napthol in the presence of an oxidizing agent such as Cl₂ /Br₂ .

3. Test for sulfur

Solution contents:
40% NaOH, Lead acetate

Tested for:
Sulfur. (Cysteine, Methionine)

Theory and observations:
By boiling with NaOH, S in the amino acid is converted into NaS, which then precipitates as balck PbS with the addition of lead acetate.

4. Nitroprusside test

Solution contents:
Sodium nitroprusside, NH₃(aq)

Tested for:
Sulfhydryl group –SH. (Cysteine)

Theory and observations:
The group reacts with nitroprusside in alkaline solution and gives in a red colour complex.

5. Hopkins- Cole test

Solution contents:
Sodium nitroprusside, NH₃(aq)

Tested for:
Indol group. (Tryptophan)

Theory and observations:
The indol group reacts with glycoxilic acid in conc. Sulfuric acid to form a purple colour.

http://www.chem.boun.edu.tr/webpages/courses/chem415/CHEM415-Experiment2-Procedure.pdf

http://www.cerlabs.com/experiments/10875404480.pdf

Tests for Proteins -1

1.The Biuret Test

Solution contents:
KOH, CuSO_4, Potassium sodium tartarate

Tested for:
Peptide bonds. (Proteins and peptones give positive results)

Theory and observations:
Following compound is formed between the -C-N-C-back bone of the proteins and the copper(II) ions in the solution which results in a violet colour complex.

2. The Ninhydrin Test

Solution contents:
Ninhydrin

Tested for:
Amine group (mainly primary amine groups in proteins, peptones and amino acids)

Theory and observations:
Any primary amino acid will undergo the I^st reaction and will produce a blue colour and secondary amino group containing amino acid, proline will give a yellow/orange colour (II).

3. The Millon’s Test

Solution contents:
Mercury (Hg₂²⁺, Hg²⁺) in nitric acid

Tested for:
Phenol groups (Tyrosine)

Theory and observations:
Firstly the phenol ring is nitrated with conc. Nitric acid and the nitrated product will react with the mercury ions in the solution to give in either a deep red colour solution or precipitate. [Hg²⁺](Some tyrosine containing proteins will give in a white precipitate [Hg₂²⁺] which would turn red when heated.