Production of hemin crystals.

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.

Isolation of deoxyribonucleoproteins from the spleen.

Grind 5 g of spleen with an equal amount of sand in a mortar, add 5 ml of chilled 2 M solution of sodium chloride, and then gradually in small portions 25 ml of cold 1 M sodium chloride solution. Continue rubbing for 10-15 minutes in a mortar cooled with ice. Transfer the resulting mass into centrifuge tubes and centrifuge for 15 min. Measure the volume of supernatant and pour it by a thin stream into sixfold volume of water, slowly stirring the liquid with a wooden stick. Nucleoprotein filaments precipitate. If there is a flocculent precipitate, leave it for some minutes, and centrifuge the liquid.

Use sediment to do test on DNA.

Reaction with diphenylamine.

Deoxyribonucleic acid is detected by its reaction with diphenylamine. Transfer a bit of DNA sediment in the test tube and dissolve it in 2 ml of 0.4% sodium hydroxide solution. Add an equal volume of diphenylamine. Heat the mixture in boiling water bath for about 10-15 minutes.

Chromoproteins.

The representative of chromoproteins is a protein of mammals erythrocytes - hemoglobin. Its protein part is globin and non-protein is heme (protoporphyrin, linked to iron atom).

Benzidine test for hemin group of hemoglobin.

Benzidine test is used in clinical and forensic investigations for the detection of small quantities of blood in biological objects.

Pour 0.5 ml of highly diluted blood in the test tube and boil for several minutes, then cool. Add 0.5 ml of benzidine solution and 5 drops of 3% hydrogen peroxide solution. The reaction of hemoglobin with concentrated acetic acid results in hemin, which catalyzes the oxidation of benzidine by hydrogen peroxide.

Production of hemin crystals.

When dried blood is heated with concentrated acetic acid, hemoglobin is broken down to form globin and hematin. Blood sodium chloride reacts with acetic acid and forms HCl, the latter converts hematin to hemin. Hemin forms crystals after cooling. This reaction is used in forensic medicine.

Apply a drop of blood on the microscope slide and spread it by the edge of another slide. Than dry blood above the flame (temperature should not be more than 60°С). Add 2 drops of concentrated acetic acid, put the cover glass on and heat on the flame till boiling, than cool. Examine resulting specimen using a microscope.

Observations:

Tasks

1. Cytochrome c contains 0.426% of Fe. Calculate the molecular mass of this protein.

2. Dead body was found after the fire. What simple method can be used to test when the man died – during the fire or before the fire?

3. Write formulas of carbohydrates, which are parts of DNA and RNA.

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