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EXPERIMENTS WITH ENZYMES OXIDASE AND PEROXIDASE

 

In the cells of plants and animals continuously flow through complex chemical processes. They are regulated protein substances - enzymes, which act as catalysts of chemical reactions in cells. To explore these biochemical processes require sophisticated instruments and many chemicals. However, some biochemical phenomena can be observed, as they say, and to the naked eye.

Let's start with oxidative enzymes - oxidase and peroxidase. They are present in many living tissues, because the oxidation underlies the processes of respiration. But are these enzymes in different ways: oxidase oxidizes organic matter, oxygen, peroxidase for the same purpose "mined" oxygen from peroxides. Of course, the substance is slowly oxidized and without the aid of enzymes, but enzymes accelerate reactions in many thousands of times.

In the oxidation of certain substances, for example, phenol and hydroquinone, are colored reaction products. The coloration suggests that the enzyme worked. And color intensity allows you to judge the amount of oxidation products. If the color does not appear, it means that the enzyme is inactive. It can happen too acidic or too alkaline, or if there are no suppliers of oxygen or in the presence of harmful enzymes substances - the so-called enzyme inhibitors.

After this short introduction - yourself experiments. You will need: cabbage stalks, Apple, potato with sprouts, onion with roots germinated in the dark. Reagents will serve cold boiled, and even better distilled water, hydroquinone (from the store, and drugstore hydrogen peroxide. Please be also a grater for vegetables, water bath, test tubes or bottles from under penicillin, clean pipettes and gauze or white cloth.

Begin research with cabbage juice. Slice cabbage stalk, about 20 grams, grind grated, the resulting slurry squeeze through two layers of gauze or a single layer of fabric, collect juice in a glass and dilute with water ten times. Immediately warn: in the study of other plant objects juice should be diluted no more than two or three times.

Six clean, dry test tubes or vials number. In test tube No. 1, 2, 3 and 4 pour 1 ml of the diluted cabbage juice. Tubes 1 and 2 set for destruction (inactivation) of enzymes for five minutes in a boiling water bath, then cool to room temperature. In tubes 5 and 6 instead of juice pour 1 ml of water.

All six test tubes, add a little pinch of hydroquinone. Then in tubes 1, 3 and 5 pour five drops of water, and the tubes 2, 4 and 6 - five drops of hydrogen peroxide. The contents of each tube thoroughly mix.

After 10-15 minutes, you can already see the results of experience. We strongly recommend to record them in a table. Make a table of the number of tubes and the composition of the mixture in each of them, in the column against each mix mark, have you changed the color in the course of the experience, and if so, how. The following graph make a conclusion - any oxidation.

When the whole table is filled, try to analyze the obtained results. To do this, consider these questions.

Can hydrogen peroxide to oxidize the hydroquinone in the absence of cabbage juice?

Oxidized whether hydroquinone under the action of cabbage juice without hydrogen peroxide?

Does the activity of enzymes in the juice after boiling?

What are oxidative enzymes contained in cabbage juice - oxidase or peroxidase?

However, based on experience with plants of the same species it is still too early to draw final conclusions. Therefore, give the same experiments with potatoes and sprouts, with the pulp of apples, with fleshy scales of the bulb, and its concem and sheets ("feathers"). Note: in these cases the resulting juice should be diluted with water 2-3 times.

When all the experiments are done, you can determine which of the investigated materials oxidative enzymes active. Do you think that can simultaneously be present in plant tissues oxidase and peroxidase?

Try to make your own conclusion without looking at the explanation. And when conclusions are made, check whether they are correct.

The first conclusion. Hydrogen peroxide can gradually oxidize the hydroquinone and without juice: in test tubes 5 and 6 slowly appears pink. Hence, the enzyme is optional for the reaction. Like all catalysts, enzymes are only accelerate the beginning of the reaction many times. You noticed, of course, how quickly appeared the color in the tube 4. However, the peroxidase may not accelerate the reaction of hydroquinone with oxygen (the color in the test tube 3 is missing or appears very slowly).

The second conclusion. The enzyme can incapacitate even a short boiling. In vitro 2 color, almost none. Because enzymes are proteins; they are rolled up when heated in test tubes 1 and 2 protein flakes.

The third conclusion. In vitro 3 color does not appear. So, in cabbage juice contains only peroxidase, accelerating the oxidation of hydroquinone only in the presence of hydrogen peroxide. However, in experiments with potato tubers and Apple color appears, and especially quickly when you shake the bottle, when the solution is enriched with oxygen. So, in potato and Apple is oxidase (specifically, phenoloxidase), promote oxidation of hydroquinone by oxygen. Therefore, the darker the air sliced potatoes and apples - they contain substances related to hydroquinone. Oxidase also loses activity when heated. Remember, it gets dark if boiled potatoes?

Finally, the fourth conclusion. In potato and Apple are also oxidase - adding peroxide to the test tube 4 color appears more likely. But in fleshy scales Luke oxidase no. They do not darken in the air even with hydroquinone.

By the way, did you notice that oxidative enzymes are particularly active in preparing for growth or growing organs of plants in the bottom of the bulbs and roots, sprouts of potato tubers? Metabolism takes place most intensively.

So, we found that not all the environmental conditions favorable for the action of enzymes. If the high heat inactivates enzymes, it may be, they are more active at low temperature? Will check this. For experience will need an additional four glass or metal cans with a capacity of about one liter and ice or snow (about 1 kg ). The experience will put cabbage stalk.

RUB the loaf grated, juice, as before, press through a gauze or cloth and dilute with water twenty times. Number the tubes again, if the old numbering some reason erased, and pour into test tubes 1, 2, 3, and 4 to 1 ml of diluted cabbage juice, and then add hydroquinone on the tip of a knife. In tubes 5 and 6 instead of juice pour 1 ml of water and also put hydroquinone. And then place the test tube in the following way: 1 - in a jar with snow or ice; 2 - in a jar with warm water (40°C); 3 - in a jar with hot water (60°C); 4 - leave on the table at room temperature; 5 - in a jar of boiling water; 6 - leave at room temperature.

After 5 min after the start of the experiment in all test tubes, starting with the colder, add five drops of hydrogen peroxide. The mixture gently shake and note the start time of the reaction. After another 5 min, remove the tubes from the jar and write down your results in a table, about the same as last time. When the table is full, you can do data analysis.

Try to make conclusions on their own, responding first to the following questions.

Whether the reaction is accelerated oxidation at elevated temperature without the addition of enzyme?

Is it possible to say that enzymes are better at cooling?

What is the temperature most favorable for the action of peroxidases?

Why is food longer stored in the refrigerator?

Why boil milk?

Why warm-blooded animals - mammals and birds - the most developed and viable animals on Earth?

You have answered all these questions? Then our explanations.

You probably noticed that the rate of oxidation of hydroquinone and hydrogen peroxide differently at low and high temperatures. At high temperatures, the oxidation rate, of course, higher. Peroxidase facilitate interaction hydroquinone with peroxide. In the presence of the enzyme reaction takes place even at low temperatures, but the higher the temperature, the easier it is for the enzyme to activate the molecules of the reacting substances.

But we must not forget that proteins at high temperature is rolled, the reaction rate decreases. There is a concept of the optimal temperature of enzyme activity in which they are most active. For many enzymes, the temperature varies, but many enzymes, including peroxidase, have a temperature optimum of 40-50°C.

Foods deteriorate under the action of enzymes contained in them or are excreted by microorganisms. In the cold enzyme activity is reduced - that is why in the refrigerator, the food rots less.

On the top step in the evolution of rose-blooded animals that can maintain body temperature optimum for the activity of enzymes.

O. Holguín. "Experiments without explosions"
M., "Chemistry", 1986

 

Your comments:

Nikita
2011-09-14 10:40:10
Maria, when cooked, the enzymes are destroyed!
Maria
2011-09-14 10:37:59
why boiled potatoes are not able to decompose hydrogen peroxide?
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