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.

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.

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.

Test questions

1. How can you separate the mixture of amino acids in the hydrolyzate of the protein?

2. What color reactions for proteins are known to you from the course of bioorganic chemistry?

3. Which amino acid is founded in proteins with the help of Millon’s reaction? Give its formula.

4. Which amino acid is in proteins, and founds during Adamkevich’s and Schulze – Raspel’s reactions? Give its formula.

5. What caused precipitation of proteins by the action of salts of heavy metals?

6. What is meant by denaturation of protein?

7. What is the practical application of the reaction of precipitation of proteins by organic acids?

8. Why in clinical practice for the protein precipitation using nitrogen rather than sulfuric or hydrochloric acid?

Laboratory work 2. Conjugated proteins: glycoproteins and phosphoproteins

Phosphoproteins.

A typical representative of phosphoproteins is the casein of milk. Casein is decomposes into protein and phosphoric acid during the alkaline hydrolysis.

1.1. Proof of protein component’s present in the casein hydrolyzate.

To 0.5 ml of casein hydrolyzate were added 5 drops of 10% sodium hydroxide solution and drops of 1% copper sulphate solution until a blue-violet color. Positive biuretic reaction proves the protein nature of casein.

1.2. Proof of phosphoric acid’s presence in the casein hydrolyzate.

1 ml of the hydrolyzate is neutralized with 25% nitric acid (control is performed on the indicator paper) and add 0.5 ml of ammonium molybdate in nitric acid. After 5 minutes the precipitate proteins and products of incomplete hydrolysis of proteins are filtered. Add 0.5 ml of 2% solution of hydroquinone to the filtrate and leave for 5 minutes. Then dropwise add 1 ml of solution of sodium carbonate-sulphite. There is a blue coloration of the solution due to the formation of molybdenum blue.