Методические указания к практическим (семинарским) занятиям " Basis of biochemistry " Agrarian Faculty
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- Bu səhifədəki naviqasiya:
- Devices
- Qualitative reaction to vitamin В
- Qualitative reaction to vitamin С . Principle of the method
- Practical work № 3 Topic
- B. Influence of рН on activity of enzymes. Principle of a method
- C. Specificity of enzymes . Principle of a method.
- Practical work № 4 Topic
- B. Definition of activity of a catalase of blood. Principle of a method
- Practical work № 5 Topic
- B. Reaction of adrenaline with iodine Principle of the method
- C. Reaction of adrenaline with chloric iron.
Practical work № 2 Topic: Amino acids: classification, structure, stereochemistry, physical and chemical properties and classification amino acids forming proteins. Qualitative analysis of water-soluble vitamins The purpose of the work: Consider the qualitative reactions to water-soluble vitamins. Qualitative reaction to vitaminВ1 Principle of the method.In an alkaline environment thiazole cycle ofthiamine becomes unstable and easily cleaved to form reactive compounds - Thiamine-thiol. Thiamine - thiol is reacted with p-diazobenzenesulfonic acid (diazoreagent) to form a compound which colored to yellow- pink color. Devices: Rack with test tubes. Reactants: Fresh whole milk. Vitamin В1, 0,001%-aqueous solution.Sodium hydroxide, 5% solution. Solution A (0.9 g a phenyl-sulfo acid dissolved in 9 ml of conc. HCl acid in a volumetric flask of 100 ml, and after some time is adjusted to the mark with water. In a dark bottle solution is stored for a long time). Solution B (sodium nitrite), 5% solution, freshly prepared).A freshly prepared diazo reagent (in a volumetric flask of 50 ml which immersed in a container with ice, metered 1,5 ml of solution A and added in small portions to 7.5 mL of solution B. Volume of the solution adjusted to the mark with cold water; after 15 minutes the reagent is ready for use). Work description. In test tube mixed 2 ml of sodium hydroxide solution and 3 ml of a diazo reagent. Half of the resulting mixture is cast into another tube. Added to the tube 1-2ml milk. In the tube develops yellow-pink color. Qualitative reaction to vitamin В2 Principle of the method.Vitamin B2reduced by hydrogen, which is released by reacting metallic zinc with hydrochloric acid. Devices: Rack with test tubes. Pipettes. Rubber stoppers for tubes. Reactants: Vitamin B2, 0,005%-aqueous solution; hydrochloric acid, diluted (1:1), metallic zinc (granular). Work description. Pour intothe test tube 2 ml solution of vitamin B2, 1 ml of hydrochloric acid and stirred, and cast into an equal volume of another tube. In one of the tubes cast piece of zinc metal and both tubes are stoppered. In the tube with zinc discoloration of solution occurs within 5-10 min. Qualitative reaction to vitamin В6(РР) Principle of the method. Under the action of sodium hydrosulfite is restored nicotinamide (nicotinic acid) to give 1,4 - dihydro pyridine derivative, a yellow colored. Devices: Rack with test tubes. Reactants: Nicotinic acid or nicotinic acid amide (powder). Sodium bicarbonate, 10% solution.Sodium hydrosulfite (Na2S2O4 * 2H2O), a 5% solution, freshly prepared. Work description. Inthe test tube is placed a little vitamin B5 powder (nicotinic acid or nicotinamide), add 1.2 ml of sodium carbonate solution and after stirring, 2.1 ml of a solution of sodium hydrosulfite. The liquid in the tube becomes yellow. Qualitative reaction to vitamin С. Principle of the method. Ascorbic acid reduces iron in the complex ion - ferrocyanide, turning it into a hexacyanoferrate, painted in blue color (Prussian Blue). Devices: Rack with test tubes. Reactants: Potassium hexacyanoferrate (6К3[Fe(CN)6]), 1% solution; ferric chloride, 1% solution; Vitamin C, 0.5% solution. Work description. Into the test tube pour 2.3 ml of water, poured into another test tube 3.2 ml of a solution of vitamin C. In both tubes add a few drops of a potassium hexacyanoferrate solution and ferric chloride. In the tube with Vitamin C solution appears blue color. The results write to the table 2. Table 2
Questions for self-preparation of students: 1. Characteristicsof vitamins B: -B2/ riboflavin/ - В12/ cobalamin/ -В3/ pantothenic acid/ - Вс/ folic acid/ -В5/ РР, nicotinamide/ - В15/ pangamic acid/ 2. Characteristicsof vitamins С: - С / ascorbic acid/ - Р/ flavone/
The general properties of enzymes – thermolability, specificity and influence рН on activity of enzymes. The purpose of the work: To study properties of enzymes. А. Thermolability of enzymes. Principle of the method. Influence of change of temperature of the external environment on activity of enzyme of amylase of saliva is investigated. Devices: Support with test tubes; glass (50 ml); spirit-lamp; thermostat (37 ͦC); a glass with ice. Reactants: The diluted saliva (rinse a mouth with the distilled water, and then, having gained 20-25 ml of water in a mouth, bring together her in a glass); sodium chloride; 0,3% solution of chloride of sodium; the Lugol reactant (приготовление:в 100 ml of water dissolve 20 g of iodide of potassium and 101 g of iodine. For reaction with starch the received solution is dissolved with the distilled water 1:5). Work description. In three test tubes pour 2-3 ml of the diluted saliva (amylase). Saliva in a test tube 1 is boiled within 1-2 min. Then add 4-5 ml of starch to all test tubes. Test tubes 1 and 3 put in the thermostat / 37 ͦ For 10 min. The test tube 3 is immersed for 10 min. in ice. After the specified time add to all test tubes on 1 drop of a reactant of Lugol. Results of experience enter 3 thermolabilities of enzymes in the table and draw conclusions. Thermolability of enzymes Table 3
B. Influence of рН on activity of enzymes. Principle of a method. Activity of enzyme of amylase of saliva at various values of the environment is investigated. Devices: A support with test tubes. Pipettes. Thermostat /37ͦ С/. Reactants: Disubstituted format of sodium, 0,2 M solution/B/; buffer solutions (рН 5,0: To mix 515 ml of solution A from 485 ml of solution B; рН 6,8: To mix 772,5 ml of solution A from 27,5 ml of solution B). The diluted saliva (Starch, 1% solution. Lugol reactant). Work description. In three test tubes flow 2-3 ml of buffer solutions with various rn/5,0; 6,8; 8,0/. Add to all test tubes on 2-3 ml of the diluted saliva / amylase(s) on 4-5 ml of solution of starch, 10 min. in the thermostat / 37 ͦ With / mix and incubate. Then add to each test tube on 1 drop of a reactant of Lugol. Results of observation enter in table 4 showing influence рН to activity of amylase of saliva. Table 4
C. Specificity of enzymes. Principle of a method. It is investigated impacts of enzymes of amylase and sucrose on various substrata - starch and sucrose. Devices: A support with test tubes. Pipettes. Thermostat (37 ͦ C). Spirit-lamp. Reactants: Starch, 1% solution. Sucrose, 2% solution, the diluted saliva/. Sakharaza, solution / 10 g of yeast homogenize in 100 ml vody./. Lugol reactant/. Reactant of Felinga/preparation: 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.
Questions for self-preparation of students:
− Specificity, − Influence рН Wednesdays, − Colloidal properties.
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.
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.
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.
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.
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.
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.
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