Determination of the isoelectric point of casein.

In five test tubes from a pipette poured the following amounts of 0.1n. of acetic acid solution: in the first one -0.25 ml, in the second - 0.5 ml, in the third - 1.0 ml, in the fourth - 2.0 ml and in the fifth - 4.0 ml. From another pipette in the same order in each tube we add 8.75 ml, 8.5 ml, 8.0 ml, 7.0 ml and 5.0 ml of water. After adding 0.1% solution of casein in sodium acetate pH values in the resulting mixtures will be equal to 5.3, 5.0, 4.7, 4.4 and 4.1, respectively.

In each tube 1 ml of casein are poured with a pipette and observe the degree of turbidity of the solution in them. Where the maximum turbidity, pH corresponds to the isoelectric point of protein (pH 4.7).

Glycoproteins.

Gastrointestinal mucins are glycoproteins, the carbohydrate part of which is represented by mucopolysaccharides. They protect mucous membranes from chemical, mechanical and thermal stresses and digestion by proteolytic enzymes.

Isolation of mucin from the saliva.

Collect 1 ml of saliva in each of two test tubes and add to each instill of 1% acetic acid solution until the clot mucin. Mucin precipitate in the tubes gently washed with water, holding the clot with wand.

Reactions on the protein part (biuretic reaction).

To open a protein in a test tube with the precipitate of mucin 10% sodium hydroxide solution is added with stirring, to dissolve the clot and produce biuretic reaction. A positive result of the reaction shows protein nature of mucin.

Reaction on the carbohydrate group (naphtol test).

Mucin carbohydrates are exhibited by naphthol test (Podobedov-Molisch’s reaction). Hydroxymethylfurfural is formed from hexoses under the influence of sulfuric acid. At its interaction with α-naphthol colored condensation product is formed.

0.5 ml of 1% alcoholic solution of α-naphthol is added in the second test tube with the precipitate of mucin, stirred and gently layered on the wall 0.5 ml of concentrated sulfuric acid. At the liquid gradually appears violet-red ring.

2.4. Proof of carbohydrate’s presence in the egg albumin.

4 ml of a dilute solution of egg albumin is poured in the test tube, added 0.5 ml of 0.1% solution of α-naphthol, mix well. On the wall gently layered concentrated sulfuric acid. When standing at the boundary of the solution purple ring is observed.

Test questions

1. Which proteins are called conjugated?

2. How does a bond between the polypeptide chain and phosphoric acid in phosphoprotein form?

3. Why do a special role for the fetus, embryo, and newborn phosphoproteins play?

4. What is the difference between glycoproteins and proteoglycans?

5. What types of relationships exist between the carbohydrate and protein components?

6. Why glycoproteins are less sensitive to the denaturation factors than simple proteins?

Laboratory work 3. Conjugated proteins: nucleoproteins and chromoproteins

Nucleoproteins

Deoxyribonucleoproteins (DNP) are isolated from tissues rich in cell nuclei (thymus, spleen, and sperm). DNP are dissolved in an average concentration of salts, such as sodium chloride (1 M), and again precipitated at a dilution of the latter (up to 0.15 M) in the form of fibrous nucleoprotein.