Методические указания к практическим (семинарским) занятиям " Basis of biochemistry " Agrarian Faculty

Practical work № 12

Topic: Concept about a metabolism and metabolic paths

Metabolism of proteins

Purpose: Determination of ammonia in blood with Nessler's reagent.

Principle of the method. The method is based on compound of ammonia with sulfuric acid from education sulfate ammonit which with Nessler's reagent gives yellow coloration. Intensity of a painting of solution depends on concentration of ammonia in blood and is defined spectrophotometric.

Reagents and equipment

1. Nessler's reagent: 34,9 g of KI and 45,5 g of HgI2 dissolve in a small amount of bidistillirovanny water, add 112 g of KOH and bring volume to 1 l water.

2. Carbonate and bicarbonate buffer рН =10,2:

And. 6M to add K2CO3-414g water to 500 ml

Б.3М To add KHCO3-150g water to 500 ml.

For preparation of the buffer it is necessary to mix solutions and/and/.

3. Penicillinic bottles.

4. Corks with polished sticks.

5. Rotor

6. Spectrophotometer

7. 1H sulfuric acid (27,2 ml konts.h2so4 to 1 l)

Operation course. To enter 0,5 ml of the buffer to penicillinic bottles рН =10,2, to surge 1 ml of blood and to close immediately cork with a tugootshlifovanny stick which end shall be moistened 1H H2SO4 (after the ground stick was dipped in a glass with 1H sulfuric acid, it is necessary to remove excesses of acid percussion about hour glass.

To insert bottles into holes of a rotor and to rotate 60 min. Then carefully to open cork, to wash away the end of a stick of 5 ml of a reagent of Nesseler diluted (1 ml of solution of Nesseler of +5 ml of a redistillator). To fill a basin and to fotometrirovat on the spectrophotometer in case of wavelength 420 nanometers, against monitoring in which instead of 1 ml of blood water, 1 ml is poured. On a gage curve find the content of ammonia in blood in % mg.

Questions for self-preparation of students:

1. Value of proteins in feeding of animals. Nitrogenous balance.

2. Biologicheskayapolnetsennost of proteins.

3. Znamenimy and irreplaceable amino acids.

4. Digestion of proteins in digestive tract.

5. Features of digestion of proteins at the poligastrichnykh of animals.

6. Products of rotting of proteins in intestines and a way of their neutralization.

7. Dezaminiraniye of amino acids, a vosstanovitelnoyeaminirovaniye of ketoacids in tissues of animals.

8. Reactions reamination and decarboxylations of amino acids

9. Ammonia binding reaction.

10. Urea synthesis - M. V. Nentsky's theory.

11. Modern idea of urea biosynthesis (Krebs's theory)
Practical work № 13

Topic: Preparation of stand, posters about healthy lifestyles.

The exchange of water and minerals

Purpose: To study exchange of water and mineral substances in an organism of animals. To master a technique of definition of calcium in blood serum.

Principle of a method. The calcium which is contained in blood serum in the form of soluble salts is besieged shchavelevokisly ammonium.

CaCl₂+(NH₄)₂C₂O₄=CaC₂O₄+2NH₄Cl

The formed deposit of shchavelevokisly calcium is washed ammonia solution from excess of shchavelevokisly ammonium and dissolved in sulfuric acid.

CaC₂O₄+H₂SO₄= H₂C₂O₄+CaSO₄

The released oxalic acid is ottitrovyvat 0,01H KMnO₄ solution

5H₂C₂O₄+2KMnO₄+3H₂SO₄= K₂SO₄+2MnO₄+8H₂O+10CO₂

The quantity of KMnO₄ which has gone for titration of oxalic acid is equivalent to amount of calcium in serum.

Reactants and equipment:

1. (NH₄)₂C₂O₄ (saturated solution).

2. Blood serum.

3. 2% water solution of ammonia.

4. 0,01H KMnO₄ solution.

5. The centrifuge on 1500 rpm.

6. Water bath.

7. Pipettes (2 ml, 1 ml, 5 ml).

8. Microburette (2 ml).

9. Glass stick.

Work course. The centrifugal test tube is poured by 2 ml of the distilled water, 2 ml of serum of blood, 1 ml of shchavelevokisly ammonium, stir up and leave in a support for 15 min. In parallel put check experiment where instead of serum take 2 ml of the distilled water.

Further experience with serum of blood and check experiment are carried out equally.

Centrifuge test tubes of 5 min. At 1500 rpm. At the bottom of a test tube with serum the dense deposit is besieged. Liquid of test tubes is poured out. In test tubes pour 2% of NH₄OH solution on 4 ml, stir up and again centrifuge 5 minutes.

Washing by ammonia is repeated by 3 times, merging liquid after each centrifugation. After draining of the last portion of ammonia, liquid from walls is deleted with a strip of filter paper.

In both test tubes pour 2 ml 1H of H₂SO₄ solution, mix a deposit a thin glass stick and immerse in a glass with the boiling water (carefully) for 2 min.

More hotly solution is titrut in a test tube 0,01H KMnO₄ solution before not disappearing pink coloring.

Content of calcium is calculated on a formula:

X=(а-в)*0,2*100/2 мг% Са, where:

a - the number of ml of KMnO4 which has gone for titration of a test tube with serum.

b- the number of ml of KMnO4 which has gone for titration of a control test tube.

Questions for self-preparation of students:

1. Content of water in fabrics, her physiological role and exchange.

2. Pathology and regulation of water exchange.

3. Macro - and minerals, their biological role, exchange and its pathology, regulation.

4. Interrelation of mineral elements in a metabolism.

5. Biogeochemistry and problem of mineral food of animals.

LITERATURE AND INTERNET-RESOURSES
5.1 Basic literature

5.1.1 D. Hames, N. Hooper Biochemistry - Taylor & Francis e-Library, 2006

5.1.2. Graff,S. Essays in Biochemistry/ S. Graff.- [репринтноеизд-е].- New York: Book Renaissance, 2014 [1956].- 345 p.

5.1.3 Сеитов З.С. Биохимия. –Алматы: Агроуниверситет, 2000.-897 с.

5.1.4. Биологическая химия/под ред. Н.И. Ковалевской.-М.: Академия, 2009.-255 с.

5.1.5 Berg,J.M. Biochemistry /Jeremy M. Berg, John L. Tymoczko, LubertStryer; with Gregory J. Gatto, Jr.-International seventh edition/ J.M. Berg.- New York: W.H. Freeman and Company, 2012.- 1098 с.

5.1.6 Voet,D. Biochemistry /Donald Voet, Judith G. Voet.-4-th edition/ D. Voet.- UnitedStatesofAmerica: JohnWiley&Sons, Inc, 2011.- 1428 с.

5.1.7 Кайрханов К.К. Методические указания к лабораторным занятиям по биохимии /издание третье, переработанное и дополненное/ Семипалатинск, ЦНТИ, 2003.

5.2 Additionalliterature

5.2.1 Березов Т.Т., Коровкин Б.Ф., Биохимия. -Москва: 2000.-520 с.

5.2.2 Строев Е.А. Биологическая химия. -М.: 1986.-480 с.

5.2.3 Николаев А.Я. Биохимия. -Москва: 1989.-495 с.

5.2.4 Строев Е. А., Макарова А. А., Практикум по биологической химии. -М.: «Высшая школа», 1986. -231с.
5.3 Internet-resources

5.3.1 Подред. Е.С. Северина. Биохимия: Учеб. для вузов, 2003. 779 с.

http://biochemistry.ru/biohimija_severina/B5873Content.html

5.3.2 Ridgway N., McLeod R., Vance J.E., Vance D. Biochemistry of Lipids, Lipoproteins and Membranes, 624 pages. Elsevier, 2012.

http://www.elsevier.com/books/biochemistry-of-lipids-lipoproteins-and-membranes/ridgway/978-0-444-53219-0

5.3.3 John W. Pelley, Edward F. Goljan Rapid Review Biochemistry//Chapter 3 – Membrane biochemistry and Signal Transduction, 208 pages. Elsevier, 2011.

http://www.us.elsevierhealth.com/biochemistry/rapid-review-biochemistry- paperback/9780323068871/