Учебно-методический комплекс дисциплины " Basis of biochemistry " Для специальности

Solution 1. 31,65 g of not removed crystals of a copper vitriol (CuSO4·5 H2O) dissolve in a flask on 500 ml at first in a small amount of the distilled water, and after crystals bring to a tag.

Solution 2. 173 g of segnetovy salt (potassium – sodium – tartrate) Dissolve in a flask on 500 ml in a small amount of the distilled water, add 62,5 g of the sodium hydroxide dissolved in 100 ml of the distilled water well mix and bring to a tag. Before the use solutions 1 and 2 mix in different volumes.

Work description. In test tubes 1 and 2 pour 4-5 ml of solution of starch, in test tubes 3 and 4 – on 4-5 ml of solution of sucrose. To test tubes 1 and 3 add 2-3 ml of the diluted saliva (amylase), to test tubes 2 and 4 on 2-3 ml of solution of sucrose. Contents of test tubes 10 min. in the thermostat mix and incubate (37· C). Then 1 and 2 add to test tubes on 1 drop of a reactant of Lugol, in test tubes 3 and 4 – on 1-2 ml of a reactant of Felinga and heat. Observation is written down in the table of the 5th specificity of enzymes of amylase and sucrose.
Specificity of enzymes.

Table 5.

№	Substrate	Enzyme	Incubation	Coloring with iodine	Reaction of Feling
1.	Starch	Amylase	10 min. 37ͦ С
2.	Starch	Sakharaza	10 min. 37ͦ С
3.	Starch	Amylase	10 min. 37ͦ С
4.	Starch	Sakharaza	10 min. 37ͦ С

Questions for self-preparation of students:

1. 
   The general concept about the fermetakh.
   
2. 
   Short story of development of the doctrine about enzymes.
   
3. 
   Nomenclature and classification.
   
4. 
   The most important methods of definition, receiving and cleaning.
   
5. 
   Main properties of enzymes:
   

• 
  Thermolability,
  

− Specificity,

− Influence рН Wednesdays,

− Colloidal properties.
Practical work № 4

Topic: Classification of proteins. The role of proteins in a food.

Mechanism of effect of enzymes. Influence of activators and inhibitors.

Purpose: To study the mechanism of effect of enzymes, their activators and inhibitors.

А. Influence of activators and inhibitors.

Principle of a method. In 2 test tubes pour 4-5 ml of solution of starch, 1 add 1-2 ml of solution of sulfate of copper to a test tube. In both test tubes flow 1-2 ml of the diluted saliva, mix contents of test tubes and test tubes incubate 10 min. in the thermostat /37ͦ С /. Then add to both test tubes on 1 drop of a reactant of Lugol. Observations enter in table 6 showing influence of chloride of sodium and sulfate of copper on activity of amylase.
Influence of activators and inhibitors.

Table 6.

№	Enzyme	Effector	Substrate	Incubation	Coloring with iodine
1.	Amylase	NaСl	Starch	10 min. 37ͦ С
2.	Amylase	CuSO4	Starch	10 min. 37ͦ С

B. Definition of activity of a catalase of blood.

Principle of a method. An indicator of activity of enzyme is releases of the molecular oxygen which is formed at hydrogen peroxide splitting by a blood catalase.

Devices: A support with test tubes. Pipettes. Spirit-lamps.

Reactants: Blood integral, tsitratny. Hydrogen peroxide, 3% solution.

Work description. In two test tubes pour 2-3 ml of the distilled water, add 1-2 drops of blood and 1/kontrol/heat contents of a test tube to boiling for enzyme destruction. In a test tube 2/experience / enzyme are active. Add 1 ml of solution of peroxide of hydrogen to both test tubes. In a test tube 2 observe rough release of oxygen. Results of experience enter in table 8.
Definition of activity of a catalase of blood.

Table 8.

№	Reactants:	Observed coloring	Conclusions
1
2

Questions for self-preparation of students:

1. Chemical nature and structure of enzymes:

− Coenzymes,

− Active centers,

− Substratny center,

− Allosterichesky center

2. Functions of enzymes and their localization in a cage (kompartmentalization).

3. Multifermental systems. Isoenzymes.

4. The factors influencing the speed of enzymatic reactions:

− Concentration of a substratum and enzyme,

− Activators and inhibitors,

− Konkuretny and noncompetitive inhibitors,

− nature of packaging of polypeptide chains.

5. Mechanism of effects of enzymes:

− Theory of the intermediate relations,

− the Adsorptive theory.
Practical work № 5

Topic: The nomenclature and classification of ferments. Frame and catalytic properties of ferments.

Qualitative reactions to insulin and adrenaline.

Purpose: To study qualitative reactions of hormones.

А. Qualitative reactions to insulin

Biuret reaction.

Principle of a method. In alkaline solution at copper sulfate addition such substances as a biuret, an oxalamide, polypeptides and proteins, form the complex salts painted in violet color.

Devices: A support with test tubes. Pipettes. Spirit-lamps.

Reactants: Insulin, solution / patent drug of insulin in ampoules with concentration of 40 PIECES/ml dilute with a double amount of water. Sodium hydroxide, 10% solution. Copper sulfate, 1% solution.

Work description. Add the peer volume of solution of sodium hydroxide and 1-2 drops of copper sulfate solution to 1-2 ml of solution of insulin. In a test tube the violet staining appears.

Reactions with the concentrated nitric acid.

Devices: A support with test tubes. Pipettes. Spirit-lamps.

Reactants: Insulin, solution / patent drug of insulin in ampoules with concentration of 40 PIECES/ml dilute with double amount of water/. The concentrated nitric acid.

Work description. In a test tube pour 1 ml of the concentrated nitric acid and strictly on a wall flow 1 ml of solution of insulin on drops. On the section of two liquids accurately expressed ring is formed. To write down the observed phenomena in table 9.

Table 9.

№	Reactants	Observed coloring	Principle of the method

B. Reaction of adrenaline with iodine

Principle of the method. In case of adrenaline solution heating with iodine the adrenaline oxidation products painted in red color are formed.

Devices: A support with test tubes. Pipettes. Spirit-lamps.

Reactants: Adrenaline, solution / patent medicine of adrenaline in ampoules with concentration 1:1000 dilute with double amount of water/. Iodine, 0,1 N spirtovy solution.

Work description. In one test tube pour 1-3 ml of water, and in another - 1-2 ml of solution of adrenaline. Add 2 drops of solution of iodine to both test tubes and slightly warm up. In a test tube with adrenaline red coloring appears. To enter results in table 10.

Reaction of adrenaline with iodine.

Table 10.

№	Reactants	Observed coloring	Principle of the method

C. Reaction of adrenaline with chloric iron.

Principle of a method. At addition to solution of adrenaline of solution of chloric iron green coloring which emergence is caused by existence of the rest of pyrocatechin in an adrenaline molecule develops.

Devices: Devices: A support with test tubes. Pipettes. Spirit-lamps.

Reactants: Solution adrenaline. Chloric iron, 1% solution.

Work description. In one test tube pour 1-2 ml of water, in another-1-2 ml of solution of adrenaline. Add 2-3 drops of solution of chloric iron to both test tubes. In a test tube with adrenaline green coloring appears. To issue observation in table 11.

Reaction of adrenaline with iron.

Table 11.

№	Reactants	Observed coloring	Principle of the method

Practical work № 6

Topic: Temperature effect, рН, concentration of ferment and substrate for speed of enzymatic reactions. Regulation of activity of ferments.

Metabolism and energy. Biological oxidation.

Purpose: To get acquainted with method of studying of a metabolism and energy. To reveal understanding students of the main questions of biological oxidation.

Questions for self-preparation of students:

1. A concept about a metabolism and energy.

2. A metabolism and energies - the single interconnected process of an organism.

3. Main stages of a metabolism.

4. Methods of studying of a metabolism on stages.

5. Biological oxidation:

− Bach's Theory,

− Palladin-Wiland's Theory,

− Modern representation about biological an okislenii.dykhatelny chain

− Oxidizing phosphorylation and free oxidation. Coefficient Р/О

− Overall effectiveness of accumulation of energy in case of an okislitelnomfosforilrovaniye.

Practical work № 7

Topic: The definition, constitution and classification of vitamins and their role in enzymatic reactions and in exchange processes.

Metabolism of carbohydrates (anaerobic breakdown).

Purpose: To study the mechanism of intermediate exchange of carbohydrates. Refraktometric determination of dairy sugar in milk.

Principle of method. The method is based on dependence of index of refraction of a beam on content in solution of dairy sugar. For this purpose from milk previously emit serum in which determine index of refraction. Determination is made at a fixed temperature.

Reactants and equipment:

1. Refractometer

2. A dropper from 4% solution of chloride calcium

3. The boiling bath

4. Piece test tubes-2

5. Pipettes on 5 ml

6. Funnel

7. Filters

Work description.

1. To measure 5 ml of the researched milk in a test tube, to add 5-6 drops of 4% of solution of chloride calcium.

2. A test tube with mix to mix and deliver in the boiling bath for 10 min.

3. To take out a test tube from a bath and to cool it to room temperature.

4. To filter content of a test tube via the dry filter.

5. To apply a drop of the received filtrate on the lower prism of the refractometer.

6. To lower the upper prism of the refractometer and to make counting on a scale of index of refraction.

7. Using the size of index of refraction, to determine percentage of dairy sugar by the table.

Table 18

Refractivity	Dairy sugar in %	Refractivity	Dairy sugar in %
1,3400	3,52	1,3420	4,49
1,3401	3,57	1,3421	4,54
1,3402	3,61	1,3422	4,59
1,3403	3,65	1,3423	4,64
1,3404	3,69	1,3424	4,69
1,3405	3,73	1,3425	4,74
1,3406	3,77	1,3426	4,79
1,3407	3,82	1,3427	4,84
1,3408	3,87	1,3428	4,89
1,3409	3,93	1,3429	4,95
1,3410	3,98	1,3430	5,00
1,3411	4,03	1,3431	5,05
1,3412	4,08	1,3432	5,10
1,3413	4,13	1,3433	5,15
1,3414	4,18	1,3434	5,20
1,3415	4,23	1,3435	5,25
1,3416	4,28	1,3436	5,30
1,3417	4,33	1,3437	5,35
1,3418	4,36	1,3438	5,40
1,3419	4,44	1,3439	5,45

Questions for self-preparation of students:

1. Value of carbohydrates in a metabolism and energy of an animal organism.

2. Mono - di - polysaccharides, their structure, distribution and a role.

3. Digestion and absorption of carbohydrates in digestive tract of animals. Cellulose splitting.

4. Education and disintegration of a glycogen in muscles and a liver. Blood sugar.

5. Features of transformation of carbohydrates in a hem of ruminant.

6. Intermediate exchange of carbohydrates. Glycolysis and гликогенолиз. Similarity and distinction of these processes.

7. Anaerobic splitting of carbohydrates.

8. Similarity and distinction between glycolysis and spirit fermentation.

9. Balance of energy.
Practical work № 8

Topic: Classification of carbohydrates and their most important reactions. Disaccharides and polysaccharides: lactose, maltose, sucrose, starch, glycogen, cellulose, quinine. The role of carbohydrates in a food.

Metabolism of carbohydrates (aerobic breakdown).

Purpose: Study the mechanism of intermediate exchange of carbohydrates. Definition of glucose by Bertrán's method.

Principle of a method. The method is based on ability of free carbonyl group of glucose to restore in alkaline solution oxide medi-CuO (II) in copper protoxide – Cu₂O (I).

Reactants and equipment:

1. Liquid of Felinga

2. Sulfate iron (II) solution

3. 0,1H KMnO4 solution

4. Hot water

5. A glass on 150 ml

6. Bunsen's flask

7. Shot's filter

8. Pipettes, measured cylinders

9. Water-jet pump or Kamovsky's pump

10. Rangette

Work description. In a flask with a capacity at 150 ml pour in 20 ml of the studied solution, then add 40 ml of liquid of Felinga. After that contents of a flask are heated to boiling, and boiled exactly 3 minutes. To the received red deposit of protoxide of copper (I) filter a decantation via the glass filter in Bunsen's flask for suction. After that whenever possible, without losing a deposit of protoxide of copper (I), wash it from alkali two-three times the hot distilled water. The washed-out deposit of protoxide of copper (I) is dissolved in small (5 ml) by quantities of Fe₂(SO₄)₃ and merge on the glass filter. The filter 2-3 times small wash out quantities (2-3 ml) of Fe₂(SO₄)₃ and H₂SO₄, so that on the filter there is no copper protoxide left. Then the filter and a flask are washed out hot water. The received astvor of protoxide of copper is titrut by 0,1 N KMnO₄ solution before emergence of pink coloring. Reaction proceeds on the equation:

Cu2O+ Fe₂(SO₄)₃ + H₂SO₄ = 2 CuSO₄ +2FeSO₄ +H₂O+ 10FeSO₄ +2KMnO₄+ 8H₂SO₄ = 5Fe₂(SO₄)₂+K₂SO₄+2MnSO₄+8H₂O

The number of ml 0,1H of KMnO₄ solution, left by titration is multiplied on 6,36 and according to the table find the content of glucose in the studied solution.

Table 19

Definition of glucose by Bertrán's method

Glucose, mg	Copper, mg	Glucose, mg		Copper, mg
10	20,4	43		82,9
11	22,4	44		84,7
12	24,3	45		86,4
13	26,3	46		88,2
14	28,3	47		90,0
15	30,2	48		91,8
16	32,2	49		93,6
17	34,2	50		95,4
18	36,2	51		97,1
19	38,1	52	98,9
20	40,1	53	100,6
21	42,0	54	102,3
22	43,9	55	104,1
23	45,8	56	105,8
24	47,7	57	107,6
25	49,6	58	109,3
26	51,5	59	111,1
27	53,4	60	112,8
28	55,3	61	114,5
29	57,2	62	116,2
30	59,1	63	117,9
31	60,9	64	119,6
32	62,8	65	121,3
33	64,6	66	123,0
34	66,5	67	124,7
35	68,3	68	126,4
36	70,1	69	128,1
37	72,0	70	129,8
38	73,8	71	131,4
39	75,7	72	133,1
40	77,5	73	134,7
41	79,3	74	136,3
42	81,1	75	138,9

Questions for self-preparation of students:

1. Aerobic metabolism of lactic acid (a lactate to a piruvat and CoA acetyl.

2. Cycle trikarbonovykh of acids or Krebs's cycle, its biological value.

3. Balance of energy.

4. Regulation and pathology of carbohydrate exchange.