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ENTERTAINING EXPERIENCES AT HOME
Experiments with enzymes: oxidases and peroxidases. Chemical experiments
Entertaining experiences at home / Chemistry experiments for children In the cells of plants and animals, complex chemical processes continuously occur. They are regulated by protein substances - enzymes, which (we recall once again) play the role of catalysts for chemical reactions in cells. Complex instruments and many reagents are needed to study such biochemical processes. However, some biochemical phenomena can be observed, as they say, with the naked eye. Let's start with oxidative enzymes - oxidases and peroxidases. They are present in many living tissues because oxidation underlies the processes of respiration. But these enzymes act differently: oxidases oxidize organic substances with atmospheric oxygen, peroxidases “extract” oxygen from peroxides for the same purpose. Of course, substances are slowly oxidized even without the help of enzymes, but enzymes speed up the reaction many thousands of times. When some substances are oxidized, for example, phenol and hydroquinone, colored reaction products are formed. The appearance of color indicates that the enzyme has worked. And the color intensity allows you to judge the amount of oxidation products. If the color does not appear at all, then the enzyme is inactive. This can happen in an environment that is too acidic or too alkaline, or if no oxygen supply is available, or in the presence of enzyme-damaging substances, so-called enzyme inhibitors. After this short introduction - the experiments themselves. You will need: a cabbage stalk, an apple, a potato tuber with sprouts, an onion with roots sprouted in the dark. The reagents will be cold boiled, and even better distilled water, hydroquinone (from a photographic store) and pharmacy hydrogen peroxide. Also stock up on a vegetable grater, water bath, penicillin test tubes or bottles, clean pipettes and gauze, or a white cloth. Let's start with cabbage juice. Grind a piece of cabbage stalk, about 20 g, squeeze the resulting slurry through two layers of gauze or one layer of cloth, collect the juice in a glass and dilute with water ten times. We immediately warn you: when studying other plant objects, the juice should be diluted no more than two to three times. Number six clean, dry test tubes or vials. Pour 1 ml of diluted cabbage juice into test tubes No. 2, 3, 4 and 1. Place tubes 1 and 2 to destroy (inactivate) the enzymes for five minutes in a boiling water bath, and then cool to room temperature. Pour 5 ml of water into test tubes 6 and 1 instead of juice. To all six test tubes, add a little, at the tip of a knife, hydroquinone. Then pour five drops of water into tubes 1, 3 and 5, and five drops of hydrogen peroxide into tubes 2, 4 and 6. Mix the contents of each tube thoroughly. After ten to fifteen minutes, you can already observe the results of the experiment. We strongly advise you to write them down in the form of a table. Enter in the table the numbers of test tubes and the composition of the mixture in each of them, in the column against each mixture, mark whether the color has changed during the experiment, and if it has changed, then how. In the next column, conclude whether oxidation has occurred. When the entire table is filled, try to analyze the results. To do this, think about these questions. Can hydrogen peroxide oxidize hydroquinone in the absence of cabbage juice? Is hydroquinone oxidized by cabbage juice without hydrogen peroxide? Is enzyme activity retained in juice after boiling? What oxidative enzymes are found in cabbage juice - oxidases or peroxidases? However, based on experience with plants of the same species, it is too early to draw final conclusions. Therefore, set up the same experiments with a potato tuber and its sprouts, with the pulp of apples, with the fleshy scales of an onion, as well as with its bottom and leaves ("feathers"). We remind you: in these cases, the resulting juice should be diluted with water 2-3 times. When all the experiments have been done, it is possible to determine in which of the studied materials the oxidizing enzymes are more active. In your opinion, can oxidases and peroxidases be present simultaneously in plant tissues? Try to draw your own conclusions without looking at the explanation. And when the conclusions are made, check how correct they are. Conclusion one. Hydrogen peroxide can gradually oxidize hydroquinone even without juice: a pink color slowly appears in tubes 5 and 6. This means that the enzyme is not required for the reaction. Like all catalysts, enzymes only speed up the reaction that has begun many times over. Of course, you noticed how quickly the color appeared in tube 4. However, peroxidases cannot accelerate the reaction of hydroquinone with atmospheric oxygen (the color in tube 3 is absent or appears very slowly). Second conclusion. The enzyme can be disabled even by short-term boiling. There is practically no color in test tube 2. After all, enzymes are proteins; they coagulate when heated - protein flakes appeared in test tubes 1 and 2. Conclusion the third. No color appeared in test tube 3. This means that cabbage juice contains only peroxidases, which accelerate the oxidation of hydroquinone only in the presence of hydrogen peroxide. However, in experiments with potato tubers and apples, color appears, and especially quickly when the vial is shaken, when the solution is enriched with atmospheric oxygen. This means that there are oxidases in potatoes and apples (more specifically, phenol oxidase), which contribute to the oxidation of hydroquinone with oxygen. Therefore, cut potato tubers and apples darken in the air - they contain substances related to hydroquinone. Oxidase also loses activity when heated. Remember if boiled potatoes darken? Finally, the fourth conclusion. There are also oxidases in potatoes and apples - when peroxide is added to test tube 4, the color appears sooner. And there is no oxidase in the fleshy onion scales. They do not darken in air even with hydroquinone. By the way, have you noticed that oxidative enzymes are especially active in preparing for growth or growing plant organs - in the bottom of the bulb and its roots, in the sprouts of potato tubers? The metabolism goes there most intensively. So, we found out that not all environmental conditions are favorable for the action of enzymes. If strong heat inactivates enzymes, then maybe they are more active at low temperatures? Let's check this too. For the experiment, you will need an additional four glass or metal cans with a capacity of about one liter and ice or snow (about 1 kg). Let's experiment with a cabbage stalk. Grate the stump, squeeze the juice, as before, through gauze or cloth and dilute with water twenty times. Re-number the tubes if the old numbering has somehow been erased, and pour 1 ml of diluted cabbage juice into tubes 2, 3, 4 and 1, and then add hydroquinone at the tip of a knife. In test tubes 5 and 6, instead of juice, pour 1 ml of water and also add hydroquinone. And then arrange the test tubes as follows: 1 - in a jar of snow or ice; 2 - in a jar of warm water (40°C); 3 - in a jar of hot water (60°C); 4 - leave on the table at room temperature; 5 - in a jar of boiling water; 6 - leave at room temperature. 5 minutes after the start of the experiment, pour five drops of hydrogen peroxide into all test tubes, starting with the colder ones. Shake the mixture gently and note the reaction start time. After another 5 minutes, remove the test tubes from the jars and write down the results of the experiment in the form of a table, approximately the same as the last time. When the table is filled, you can start analyzing the received data. Try to draw your own conclusions by first answering the following questions. Does the oxidation reaction accelerate with increasing temperature without the addition of an enzyme? Is it possible to say that enzymes work better when cooled? What temperature is most favorable for the action of peroxidases? Why does food last longer in the refrigerator? Why boil milk? Why are warm-blooded animals - mammals and birds - the most developed and viable animals on Earth? Have you answered all these questions? Then - our explanations. You have probably noticed that the rate of oxidation of hydroquinone with hydrogen peroxide is not the same at low and high temperatures. At high temperatures, the rate of oxidation is naturally higher. Peroxidases facilitate the interaction of hydroquinone with peroxide. In the presence of an enzyme, the reaction proceeds even at low temperatures, but the higher the temperature, the easier it is for the enzyme to activate the molecules of the reactants. But we must not forget that proteins coagulate at high temperatures, the reaction rate decreases. There is a concept of the optimal temperature for the action of enzymes, at which they exhibit the greatest activity. For different enzymes, this temperature is not the same, but many enzymes, including peroxidases, have a temperature optimum of 40-50°C. Food products are spoiled by the action of enzymes contained in them or released by microorganisms. In the cold, enzyme activity decreases - that's why food spoils less in the refrigerator. Warm-blooded animals have risen to the upper stage of evolution, which can maintain a body temperature that is optimal for the activity of enzymes. Author: Olgin O.M.
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