2. Colour reaction of proteins.
2.1. Millon’s reaction.
Millon's reagent consists of nitrate of mercury (I) and (II) in HNO3 with admixture of HNO2. The reaction of Millon's reagent with protein due to the presence of tyrosine residue in them.
In the test tube 0.5 ml protein solution is poured and 0.5 ml of Millon's reagent. A white precipitate is formed under the action of mercury salts and nitric acid, which are the part of Millon's reagent. When heated, the precipitate is colored in pink at first, and then in a brown-red color.
2.2. Ninhydrin’s reaction.
The reaction is due to the presence of α-amino acids residues in the protein.
1 ml protein solution is poured in the test tube, 5 drops of 0.2% ninhydrin solution is added and heated. After a few minutes we observe the appearance of a blue-violet color.
+ RСОН + СО2 + NН3
3. Reactions of precipitation of proteins.
3.1. Precipitation of proteins with salts of heavy metals.
Proteins interacting with salt of heavy metals (copper, mercury, lead, etc.), denatures and form insoluble complex compounds due to adsorption of heavy metal on the surface of the protein molecule. This underlies the use of proteins as an antidote for poisoning by heavy metals.
0.5 ml of protein is poured in two test tubes. In the first one we add 2 drops of 5% copper sulphate solution, in the second - 2 drops of 5% solution of lead acetate. There is the protein precipitate in both tubes.
3.3. Precipitation of proteins with the help of concentrated mineral acids.
Precipitation of protein after it’s’ contact with concentrated mineral acids due to the dehydration of protein molecules, the formation of insoluble protein complex salts. In an excess of sulfuric and hydrochloric acids the protein precipitate is dissolved. Excess of nitric acid does not dissolve the precipitated protein. Reaction with nitric acid is used in clinical studies of urine for the presence of protein in it (Geller reaction).
1 ml of concentrated nitric acid is poured in the test tube and then by tilting the tube, equal volume of protein solution is carefully poured in a wall. There is a white precipitate protein at the boundary between two liquids. Shake the tube and add an excess of nitric acid. Precipitate doesn’t disappear. The experience is repeated with concentrated hydrochloric and sulfuric acids.
3.4. Precipitation of proteins by organic acids.
Reactions with trichloroacetic and sulfosalicylic (2-hydroxy-5-sulfobenzoic) acids are specific and sensitive. They are used in clinical laboratories for detecting protein in the urine and other biological liquids. Sulfosalicylic acid can precipitate peptides, which are breakdown products of proteins -. Trichloroacetic acid precipitates only proteins. It is used in determining non-protein (residual) nitrogen of blood, which consists of decomposition products and protein metabolism.
1 ml protein solution is poured in two test tubes. 1-2 drops of sulfosalicylic acid is added in one tube, and trichloroacetic acid in another. Precipitate the protein.