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

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.

Practical work № 9

Topic: Biomedical value of lipids. Frame and classificationof lipids. The constitution and transport properties of cellular membranes.
Carbohydrates

A. The proof of the restoring ability at glucose and absence her at fructose.

a) Trommer's reaction

Place 0.5 ml of 0.5% solution of glucose and 6-8 drops in a big test tube 2n.naoh. Then on drops add 0.2 N of CuSO4 solution, his dissolution won't stop yet. Carefully heat a test tube on a spirit-lamp. The blue not soluble deposit of hydrate of an oxide of copper in water gradually passes in yellow, and then into a red deposit of protoxide of copper. Do similar experience with 0.5% solution of fructose.

B. Proof of existence of a cyclic form of glucose in solution

Bring 6 drops of solution of glucose and 3 drops of solution of fuksinosernisty acid in a test tube. To stir up contents.

The pink coloring characteristic of aldehydes doesn't arise. Explain the received result.

C. Discovery of fructose (river of Selivanov).

In the first test tube bring 10 drops of 0.5% solution of fructose, in the second test tube-10 of drops of 0.5% solution of glucose. Add to both test tubes in equal volumes a freshly cooked reactant of Selivanov (0.5% solution of resorcin in 20% hydrochloric acid). Carefully heat on a spirit-lamp. In a test tube with fructose gradually there is a red coloring.

D. The proof of lack of the restoring ability of sucrose.

Bring 0.5 ml of 0.5% solution of sucrose and 6-8 drops in a test tube 2n.rastvora a caustic natr. Then on drops add 0.2 N of solution of sulfate of copper (II), his dissolution won't stop yet. Will carefully heat a test tube on a spirit-lamp. The blue deposit doesn't pass in yellow, and then – in red that proves lack of the restoring ability at sucrose.

E. The proof of the restoring ability of lactose.

Bring 0.5 ml of 1% solution of lactose and 6-8 drops of 2 N of solution of a caustic natr in a test tube. Then on drops add 0.2n. solution of sulfate of copper (II) before full dissolution. Carefully heat on a spirit-lamp: the blue deposit of hydrate of an oxide of copper passes into yellow-orange.

Practical work № 10

Topic: Classification of hormones

Metabolism of lipids

Purpose: To study transformation of lipids in an organism of animals. Definition of iodic number of fat across Margoshes

Principle of the method. The Iodny number of fat is amount of iodine (in) which can join 100 g of fat, it shows degree of nonsaturation of fat. The method is based on reaction of accession of halogens to the remains of the nonlimiting fatty acids which are a part of fat.

Reactants and equipment:

1. Fat

2. Ethanol

3. 5% spirit solution of iodine

4. 1% solution of starch

5. 0,1 N solution of hyposulphite of sodium (Na2S2O3)

6. A flask with a cover on 300 ml-2 of piece

7. The burette for titration-1 of piece.

8. The cylinder on 200 ml-1 of piece

9. A pipette on 10 ml-2 of piece

10. Water bath

11. Scales

Work description.

1. A fat hinge plate (0,1-,12 g) place a flask and dissolve in 10 ml of ethyl alcohol. The flask is placed in a water bath (50-60 °C) for fat dissolution (to heat to an ischeznovaniye of drops of fat). At the same time blind experience becomes for what in other flask pour only 10 ml of ethyl alcohol. Further, with this flask perform the same operations that from skilled.

2. In both flasks add 5% of spirit solution of iodine on 5 ml, well stir up and flow 200 ml of warm water (20-30 °C). Flasks close a cover, stir up and leave to stand for 5 min.

3. Excess of iodine in both flasks is ottitrovyvat by 0,1 N hyposulphite solution. As the indicator add 10-12 drops of 1% of solution of starch. Titration will see to total disappearance of blue coloring.

4. The iodic number of the studied fat is determined by a formula:

X = (and - in) * 0,0127*100/C, where

H-iodine number of fat,

and - the number of ml of hyposulphite which has gone for titration of blind experience

in - the number of ml of hyposulphate which has gone for titration of a flask with fat

Fat S-hinge plate in, 0,0127 amount of iodine, corresponding 1 ml 0,1 N of solution of hyposulphite.

Questions for self-preparation of students:

1. Lipids. Neutral fats. Chemical structure, properties and role.

2. Saturated and nonsaturated fatty acids.

3. Zhiropodobny substances. Letsitina, kefalina, sterida, their value and formulas.

4. Value of fats in a metabolism and energy in an animal organism.

5. Digestion and absorption of fats in digestive tract.

6. A role of gastric acids in these processes.

7. Synthesis and adjournment of fats in fabrics of an animal organism.

8. Oxidizing disintegration of fats in fabrics:

− glycerin Oxidation,

− Oxidation of fatty acids.

9. Formation of ketone bodies.

10. Power balance.

11. Transformation of fosfatid, sterid and sterols.

Practical work № 11

Topic: Nucleicacids

Proteins. Physico-chemical properties.

Color reactions to proteins.

Principle of the method. Color reactions are reactions to structural elements of protein - peptide communication, on various amino acids in a protein molecule.

Devices: Test tubes. Water bath or spirit-lamp.

Reactants: Protein solution. NaOH, 20% rastvor.naoh, 30% solution. Sulfate copper, 1% solution. The concentrated nitric acid. Acetic lead 5% solution. Solution of an ingidrin, 0,5%.

1. Biuretny reaction. This reaction is the general reaction to proteins. She points to existence in a molecule of protein of peptide communications - CO-NH-. To 1 ml of solution of protein add the equal volume of 20% of NaOH and 3-4 drops of 1% of solution of sulfate CuSO4 copper, and mix. Liquid is painted in violet color. Coloring of liquid depends on the number of peptide communications. All proteins give this reaction.

2. Xanthoproteic reaction. By means of this reaction open at presence to a molecule of protein of cyclic amino acids - Thyrosinum, a tryptophan and phenylalanine.

To 1 ml of solution of protein add 0,5 ml конц. nitric acid, heat to boiling within 1-2 minutes. At the same time the formed deposit of the overthrown protein turns yellow, / there is a reaction of a nitridation of a benzolnogokolets of aromatic amino acids to formation of nitrobonds of yellow color that indicates presence of cyclic amino acids/.

3. Reaction Fouling. By means of this reaction open presence at a protein molecule sulfur of the containing amino acids - Cysteinum, cystine and methionine.

Take 1-2 ml of acetic lead in a test tube and on drops add solution of diabrotic sodium before dissolution of the formed deposit of hydrate of an oxide of lead. Flow several (4-5) drops of protein and gradually heat an admixture. Observe gradual darkening of solution.

4. Ninhydrin reaction. In a test tube would flow 5-6 drops of solution of solution of an ingidrin. Bring to boiling. There is a violet coloring.

This reaction is characteristic of α-amino acids.

To issue conclusions on works in table 14.

Qualitative reactions to proteins.

Table 14.

№	Reactions	Observed coloring	What groups open in protein
1.	Biuretny
2.	Xanthoproteic
3.	Fouling
4.	Ninhydrin

Questions for self-preparation of students:

1. General characteristic of proteins.

2. Main functions of proteins.

3. Physical and chemical properties of proteins.

4. Methods of allocation and purification of proteins.