BIOCHEMISTRY - Laboratory Practical Guide - NAU 2015

MODULE II

ENZYMES AND METABOLIC PATHWAYS.

ENERGY METABOLISM

Laboratory Work 6

DETECTION OF ENZYME ACTIVITY IN A BIOLOGICAL FLUID. DETERMINATION OF SPECIFIC PROPERTIES OF ENZYMES AS BIOLOGICAL CATALYSTS

Objective of the work: to master the fundamental Introduction/14.html">Principles of Enzyme detection and apply them to prove the presence of an enzyme in a biological fluid. To experimentally demonstrate the specific METABOLISM/8.html">Properties of Enzymes.

Basic Theoretical Background

Enzymes are biological catalysts of protein nature that share common features with inorganic catalysts as well as possess unique specific properties. The presence of an enzyme in a biological fluid can be demonstrated by its activity. Enzymes accelerate chemical reactions in which a substrate is converted into a reaction product in one way or another. Therefore, if an enzyme is active, The amount of substrate decreases while the amount of product increases. Enzyme activity can be assessed by tracking the decrease in substrate amount or the increase in product amount.

The specific properties of enzymes include the dependence of their action on Temperature, medium pH, and the presence of modulators (or effectors), which can either enhance enzyme activity (activators) or reduce it (inhibitors). Enzyme activity also depends on Substrate Concentration. In addition, enzymes exhibit high Specificity toward the substrate whose transformation they catalyze.

As temperature decreases, enzyme activity drops, reaching a minimum at 0 °C. When the temperature rises by every 10 degrees, enzyme activity increases 2- to 4-fold until it reaches a maximum. The temperature at which the maximum rate of an enzymatic reaction is observed is called the optimum temperature. Upon further heating—for most enzymes up to 50–70 °C—the enzyme loses its properties as a biological catalyst due to thermal Denaturation of the protein molecule.

Each enzyme has a specific medium pH value at which its activity is maximal, known as the optimum pH. For most human body enzymes, this value ranges from 6.8 to 7.4. Unlike the temperature dependence curve, the curve of enzyme activity versus medium pH is symmetrical.

Enzyme activators are typically Metal Ions, but occasionally anions (e.g., chloride anions for α-amylase). Their predominant MECHANISM OF ACTION is binding to the allosteric center of the enzyme, which alters the conformation of the enzyme molecule and the spatial complementarity between the enzyme and substrate molecules. Specific non-competitive Enzyme Inhibitors can act via the exact same mechanism. Competitive Inhibitors compete with the true substrate for binding sites within the active center of the enzyme. The Effect of a competitive inhibitor can be overcome by increasing the concentration of the true substrate, as it displaces the inhibitor from the active center. Inhibition can also be non-specific, caused by any factor that disrupts the Enzyme Structure and denatures it.

As substrate concentration increases, The rate of the enzymatic reaction rises. However, upon reaching a certain substrate concentration—the saturation concentration—all substrate-binding sites in the enzyme's active center become occupied, and further increases in substrate concentration do not accelerate the reaction.

Equipment: test tube rack, pipettes, dropper bottles, porcelain mortar and pestle, funnels, incubator, Water bath, gas burner.

6.1. Action of Invertase (Sucrase)

Materials and Reagents: invertase preparation (5 g of baker's Yeast ground in a porcelain mortar with 2–3 ml of distilled water and a small amount of Glass sand, mixed with 8–10 ml of water, and filtered through cotton wool), 2% sucrose solution, Fehling's reagent (see Laboratory Work 2).

Procedure

Pipette 1 ml of the enzyme preparation into a test tube, add 3 ml of sucrose solution, mix thoroughly, and place in an incubator at 38 °C. Invertase catalyzes the Hydrolysis of sucrose into glucose and fructose:

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The resulting glucose is identified using Fehling's reaction. To do this, after 15 minutes, add 2 ml of Fehling's reagent to the test tube, mix, and heat to a boil. A red precipitate of copper(I) oxide is formed. Sucrose contains no free aldehyde group and therefore lacks reducing properties.

Record the research results in Table 6.1.

Table 6.1

Determination of Invertase Activity

6.2. Action of Amylase

Materials and Reagents: starch solution (0.2%), 0.1% iodine solution in 0.2% potassium iodide solution, saliva solution containing the enzyme.

Preparation of saliva solution — rinse the Mouth two or three times with water, then use a cylinder to measure 50 mL of distilled water and rinse the mouth with it several times for 3—5 min. Filter the collected liquid through cotton wool. The filtrate is used as the enzyme source.

Procedure

Pour 5 mL of starch paste into each of two test tubes. Add 0.5 mL of saliva solution containing amylase to one of them, mix well, and let stand at room temperature. Amylase is an enzyme that catalyzes the hydrolysis of the α-1,4-glycosidic bond in starch and Glycogen into intermediate products known as dextrins. Starch (due to amylose) forms blue-colored compounds with iodine, while dextrins form violet, reddish-brown, or yellow colors. The end product of starch hydrolysis is glucose.

After 15 min, add five drops of iodine solution to both test tubes. In the test tube with salivary amylase, the solution decolorizes within 1 hour, whereas in the control test tube without amylase, the color of the solution remains unchanged, i.e., blue-violet.

6.3. Effect of Activators and Inhibitors on Amylase Activity

Materials and Reagents: saliva solution, 0.5% starch solution, 0.1% iodine solution in 0.2% potassium iodide solution,
1% NaCl solution, 1% CuSO₄ solution.

Procedure

Prepare three test tubes. Pour 2.5 mL of water into the first, 2 mL of water and 0.5 mL of NaCl solution into the second, and 2 mL of water and 0.5 mL of CuSO₄ solution into the third. Add 2.5 mL of saliva solution to all test tubes, mix, add 2.5 mL of starch solution to each, mix again, and place them in a thermostat at 38 °C. NaCl acts as an amylase activator, while CuSO₄ acts as an inhibitor. The effect of these substances on amylase activity is determined by the degree of starch hydrolysis catalyzed by the enzyme in the presence of NaCl and CuSO₄.

After 5 min, add five drops of iodine solution to each tube. The liquid in the first test tube turns violet or red, In the second — red or yellow, and in the third — blue.

6.4. Investigation of the Effect of High Temperatures on The activity of Salivary α—Amylase

Materials and reagents: starch solution (0.2%), 0.1% iodine solution in 0.2% potassium iodide solution, Fehling's reagent (see Laboratory Work 2), saliva solution containing the enzyme.

Procedure

The Effect of temperature on salivary amylase activity is demonstrated by Changes in the rate of starch hydrolysis by the enzyme under various temperature conditions. Add 1 ml of 2-fold diluted saliva to each of two test tubes, and 1 ml of similarly diluted saliva that has been pre-boiled for 2 min to two other test tubes. Add 2 ml of 0.2% starch solution to all four test tubes and place them in a thermostat at t° = 37 °C for 15 min.

The extent of starch Digestion is determined using the iodine test (by the decrease in substrate concentration) or Fehling's test (by the increase in reaction product). Following incubation, perform the iodine test by adding 1–2 drops of 0.1% iodine in 0.2% potassium iodide to two test tubes (one with unboiled saliva and one with boiled saliva), and Fehling's test by adding 2 ml of Fehling's reagent to the other two test tubes (unboiled and boiled saliva), mixing, and heating to boiling. Observe the color change in the iodine test and precipitate formation in Fehling's test.

Record the experimental results in Table 6.2:

Table 6.2

Determination of salivary α-amylase activity

6.5. Effect of pH on Amylase Activity

Materials and reagents: saliva solution, 1% starch solution, 0.1% iodine solution in 0.2% potassium iodide solution, phosphate buffers with various pH values, total buffer volume of 100 ml (Table 6.3).

Table 6.3

Composition of phosphate buffer, ml

Procedure

The effect of pH on amylase activity is determined by changes in the color intensity of the starch-iodine solution.

Pour 2 mL of Buffer solutions with different pH values into seven test tubes. Then add 5 mL of starch solution and 1 mL of saliva solution to each. Mix the Contents of the tubes and place them in a thermostat at 38 °C for 10 min. After incubation, add five drops of iodine solution to all test tubes, mix, and observe the color intensity. For clarity, you can add a few milliliters of distilled water to each test tube and mix.

The optimal pH for salivary amylase is 6,8; enzyme activity decreases in both acidic and alkaline environments.

Processing of Experimental Data

Construct a diagram illustrating the enzyme-substrate interaction during the reaction. How can the presence of an enzyme be detected in a biological fluid? Explain using the Examples of sucrase and amylase.

How can the specific properties of enzymes be demonstrated experimentally? Construct a table presenting experimental Evidence for the specific properties of amylase.

Selection/5.html">Control Questions and Tasks

1. Name the primary Methods for detecting enzymes in biological fluids, illustrating your answer with Methods for determining salivary amylase activity.

2. State the common properties shared by enzymes and inorganic catalysts, as well as the specific properties of enzymes.

3. Identify the functionally active regions of an enzyme molecule.

4. Which enzymes are referred to as allosteric?

5. Plot graphs showing the dependence of salivary amylase activity on temperature and pH.

6. How does the rate of an enzymatic reaction depend on Enzyme Concentration and substrate concentration? Plot the corresponding graphs.

7. Which constant characterizes the affinity between an enzyme and its substrate? What is its numerical value equal to?

8. Why do activators increase enzymatic activity?

9. How are enzyme inhibitors classified? Explain their mechanism of action.

10. What causes non-specific Enzyme Inhibition?

11. Explain The Mechanism of the inhibitory effect of copper sulfate on enzyme activity. What type of inhibitor does it belong to?

12. Explain the mechanism by which boiling affects enzyme activity. Suggest alternative methods for amylase inactivation.

13. Define the main Units of Measurement for enzymatic activity.

14. What is specific enzyme activity? Why is it determined?

References: [1; 4—7].