BIOCHEMISTRY - Main Regulators and Biological Fluids of the Human Body - 2016

1. ENZYMES

1.7. Practical Part. Investigation of Enzyme Properties

Aim of the work - to study the METABOLISM/8.html">Properties of Enzymes and the Specific features of biochemical reactions proceeding under the action of enzymes.

Experiment 1. Enzymatic Hydrolysis of Starch

Hydrolysis is used to study the composition of substances. Hydrolysis can be: 1) acidic, 2) alkaline, 3) enzymatic. In the first two cases, the process proceeds at an elevated Temperature, whereas in the latter case, it occurs at human body temperature.

The enzyme that hydrolyzes starch into its constituent parts (dextrins, maltose, glucose) is salivary amylase. The results of the experiment are evaluated using color reactions: Trommer's test and the iodine-starch test.

Unhydrolyzed starch gives a blue coloration with an iodine solution (I₂) (positive reaction) and a negative Trommer's test, as it lacks reducing properties.

Conversely, the products of starch hydrolysis (maltose and glucose) do not react with the iodine solution (negative reaction), but they do yield a positive Trommer's test.

Take two clean, dry test tubes and number them. Add 10 drops of a 1% starch solution to both test tubes. Add 4 drops of Water to one of them (control), and 4 drops of a 1:5 saliva dilution to the second one. Mix the contents and place them in an incubator at 37 °С for 15 minutes.

Then, check whether starch hydrolysis has occurred in each of the test tubes. To do this, take two 4-drop aliquots of the test substance from the first tube, place them into two new test tubes, and perform two respective reactions in these tubes: the iodine-starch test in one and Trommer's test in the other.

Perform a similar Procedure with the Contents of the second test tube. When preparing the report, record the experimental results in a table designed According to the template in Table 1.

Class="center">Table 1

Experimental Results

1. Iodine-starch test. Add 1 - 2 drops of a 1% iodine solution (I₂) in a 2% potassium iodide (KI) solution to the obtained solution (hydrolysate). Note The change in the color of the test tube contents.

2. Trommer's test. To the obtained solution (hydrolysate), first add 1 ml of a 10% sodium hydroxide (NaOH) solution to create an alkaline medium, and then add 1 ml of a 5% copper sulfate (CuSO₄) solution and heat in a boiling water bath. Is a color change observed? Will Trommer's test be positive?

Draw a Conclusion as to whether starch hydrolysis has occurred. What was formed As a result of hydrolysis? Include a step-by-step scheme of the Enzymatic hydrolysis of starch in the report.

Experiment 2. Effect of temperature on Enzyme Activity

Enzymes are highly sensitive to temperature and exhibit their highest activity at its optimal value, which for human enzymes ranges from 35 - 45 °С. At temperatures exceeding 50 °С, enzyme activity drops and subsequent inactivation occurs because The Structure of the enzyme's active center is disrupted, preventing its binding to the substrate.

Take two clean, dry test tubes and number them. Add 10 drops of a 1% starch solution to both test tubes. Then, add 5 drops of a 1:5 saliva dilution to one test tube, and an equal amount of pre-boiled saliva (for 10 minutes, so that the amylase is inactivated) to the other. Shake both test tubes and place them in an incubator set at 37 °С for 15 minutes, after which check whether starch hydrolysis has occurred in each of the test tubes. To do this, perform the iodine solution test (iodine-starch test) and Trommer's test with the contents of each test tube. Record the experimental results in Table 2.

Experimental Results

In the report, indicate in which test tube starch hydrolysis occurred and in which it did not, and explain why.

Experiment 3. Effect of Medium pH on Enzyme Activity

Each enzyme has an optimal environmental pH at which it exhibits maximum activity. Changes in pH slow down or completely inhibit enzyme action because they disrupt the STRUCTURE OF THE Active Site (alterations in the reaction medium change the charge of the functional groups that make up the active site).

Pour 1 ml of distilled water into each of eight numbered test tubes. Then, add 1 ml of a 0.2% Hydrochloric acid (HCl) solution to test tube No. 1, mix thoroughly, and take 1 ml of the resulting mixture to transfer into test tube No. 2. Mix the contents of test tube No. 2, take 1 ml, and transfer it to test tube No. 3, and so on. Finally, take 1 ml from test tube No. 8 and discard it. This procedure yields various dilutions of hydrochloric acid corresponding to different environmental pH values.

Next, add 2 ml of a 1% starch solution and 1 ml of a 1:10 saliva dilution to each test tube. Shake the test tubes and place them in a thermostat at 37 °C for 15 minutes. Remove the test tubes from the thermostat, let them cool, and add 1 drop of a 1% iodine (I₂) solution in 2% potassium iodide (KI) to each tube.

In your report, describe how the color of the contents in each test tube changes, and note that complete starch hydrolysis occurred in test tubes No. 5 and No. 6, where the pH was 6.8–7.2, which is optimal for amylase activity.

Experiment 4. Effect of Activators and Inhibitors on Enzyme Activity

Certain substances can enhance enzyme action (activators), while others inhibit their activity (inhibitors). For amylase, chloride anions act as activators, whereas Bile acids serve as activators for pancreatic lipase. Copper(II) ions (Cu²⁺) act as amylase inhibitors, and cyanides and similar compounds inhibit Cytochromes (enzymes involved in Biological Oxidation).

Take three dry, clean test tubes and number them. Add 1 drop of a 3% sodium chloride (NaCl) solution to test tube No. 1, 1 drop of a 1% copper(II) sulfate (CuSO₄) solution to test tube No. 2, and 1 drop of water to test tube No. 3 (control). Then, add 10 drops of a 1:5 saliva dilution to all test tubes. Mix the contents of each test tube, add 5 drops of a 1% starch solution, and leave them at room temperature for 5 minutes. Afterward, add 1 drop of a 1% iodine (I₂) solution in 2% potassium iodide (KI) to each test tube.

In your report, indicate:

1) how the color of the solution changed in each test tube;

2) in which test tubes starch hydrolysis took place and in which it did not.

Based on your observations, draw Conclusions regarding the activating or inhibiting capacity of the tested salt solutions.

Experiment 5. Proteins as an Antidote for Heavy Metal Ions

The interaction between metal ions and proteins forms the basis for using milk as an antidote in human heavy metal poisoning.

Take two dry, clean test tubes and number them. In the experimental test tube No. 1, combine 1 ml of amylase solution, 1 ml of 1% starch solution, 1 ml of 1% lead(II) acetate ((Ac)₂Pb) solution, and milk. In the control test tube No. 2, combine 1 ml of amylase solution, 1 ml of starch solution, 1 ml of lead(II) acetate solution, and water. Shake the test tubes and keep them in a thermostat at 30 °C for 20 minutes. Add 1 drop of a 1% iodine (I₂) solution to both test tubes. Both test tubes contain lead ions (Pb²⁺) that inactivate amylase; however, in the presence of milk, amylase activity is preserved, as indicated by the absence of a blue color upon adding iodine (a negative reaction, demonstrating that starch has been hydrolyzed).

In your report, state how the solution color changed in each test tube and explain why.

Experiment 6. Detection of Catalase in Food Products

The enzyme catalase is found in certain raw foods, such as milk, meat, and potatoes. Its presence is detected by its ability to decompose hydrogen peroxide with the release of gaseous oxygen:

Take two dry, clean test tubes and number them. Add 5 drops of hydrogen peroxide (H₂O₂) to both test tubes. Add 5 drops of raw milk to the first test tube and 5 drops of boiled milk to the second. Gas evolution occurs in the first test tube, causing a glowing splint to reignite, whereas this does not happen In the second. Instead of milk, you can use small pieces of raw and boiled meat or raw and boiled potatoes.

In your report, indicate in which test tube gas evolution was observed, and draw a conclusion regarding the presence of catalase in the tested food products.

Experiment 7. Detection of Tyrosinase in Potatoes

Tyrosinase (catechol oxidase) belongs to a group of enzymes that oxidize phenols and related compounds, particularly Tyrosine. Structurally, tyrosinase is a metalloprotein containing 0.20–0.25% copper. Copper acts as an electron carrier from the substrate to atmospheric oxygen. Tyrosinase is found in many plants and Fungi, as well as in specific animal Organs and Tissues.

Under The Influence of tyrosinase, tyrosine is oxidized to a red pigment, which undergoes further oxidation to form the black pigment melanin. The conversion of the red pigment into melanin can also occur in the absence of tyrosinase, driven solely by atmospheric oxygen.

To prepare the tyrosinase enzyme extract, peel a raw potato, take 2.0–4.0 g of the outer tuber layers, cut them into small pieces, grind them in a mortar with 10 ml of distilled water, and filter the mixture through a double layer of cheesecloth.

Take two dry, clean test tubes, number them, and pour 1 ml of the obtained filtrate into each. Boil the contents of one test tube for 1–2 minutes and cool it under running tap water. Add 1 ml of a 0.1% tyrosine solution to both test tubes, mix the contents, and place them in a water bath at 37–40 °C. Periodically shake the test tubes vigorously to ensure good contact between the mixture components and air. Gradually, in the test tube containing the active enzyme, the solution darkens due to The formation of black oxidation products resembling Melanins. In the control test tube, where the enzyme has been inactivated by heat, the color of the liquid remains unchanged.

State in the report how the coloration in the test tubes changed and explain why.