Still, you raised experiments with plant tissues. Will try to experiment with enzymes from animal tissues. The object of the new research - dehydrogenase, which are involved in the processes of tissue respiration in animals. From the names of these enzymes shows that they take away from substances hydrogen atoms. As this happens, you will see a little later, but for now, prepare all the necessary experience.
This time you will need the following ingredients: 5 ml of 0.5%aqueous solution of formaldehyde (pharmacy formalin diluted with water ten times); the same amount of 0.02%aqueous solution of methylene blue (you can use a diluted blue ink); the same aqueous solution of succinic acid, carefully neutralized baking soda to stop the allocation of vials; 10 ml vaseline or vegetable oil. Still need a thermometer and objects of study - fresh cow's milk and chicken or rabbit.
First, let's see how oxidizes formaldehyde dehydrogenase fresh milk. So it became noticeable, and it took the dye solution of methylene blue: dehydrogenase Rob formaldehyde hydrogen atom transfer it on easily recovering the dye, and it will fade. And formaldehyde is oxidized up to formic acid. Discolored after recovery of the dye is easily oxidized by oxygen in the air and once again blue. Therefore, the reaction mixture will be isolated from the air by a layer of oil.
Number six test tubes. In tubes 1 and 2 pour 5 ml of fresh boiled milk, and the tube 3 is the same amount of cold boiled milk.
The water in the water bath heat to 37°C. In all three test tubes, add 0.5 ml (50 drops) of formaldehyde solution and 5 drops of a solution of methylene blue. The mixture will become blue. Into each bottle pour a little oil, to form a thin insulating layer on the surface, and place the tubes 1 and 3 in a water bath. The tube 2 leave at room temperature. Note the start time of the reaction and observe the change of color solutions. The results don't forget to write in the notebook.
As before, let us ask you leading questions. At what temperature the solution becomes colourless faster?
Does the enzyme activity in boiled milk?
What happens if you blow through a discolored solution the air?
Before checking the correctness of their conclusions, put another experience with dehydrogenases, and then consider the results of both experiments.
In the muscles of animals contains specific enzyme succinic acid dehydrogenase. It catalyzes the conversion of succinic acid to fumaric transfer taken of hydrogen at a suitable substance, for example, on methylinosine blue. This reaction proceeds without participation of oxygen, as they say, in anaerobic conditions.
Raw chicken or rabbit (about 10 g ) finely chop and mash on a plate. Gruel rinse several times with water on the gauze to remove soluble substances. The washed slurry mix with a triple volume of water to which is added salt - about a third of a teaspoon. The diluted slurry pour into the tubes 4, 5 e 6 - 5 ml each. The tube 4 soak for five minutes in a boiling water bath and cool to room temperature. Then in tubes 4 and 5 add 0.5 ml of a solution of succinic acid and 10 drops of methylene blue, and the tube 6 add 0.5 ml of water and 10 drops of dye. In each bottle, add a little oil to isolate the mixture from the air. Following the change of color, try to answer the next questions.
Restored if the dye in the presence of boiled pulp of meat?
Is there a need for this reaction succinic acid?
Similar whether the behavior of enzymes from the tissues at high temperature?
And now for the explanation. Dehydrogenase in fresh milk accelerate the oxidation reaction of formaldehyde (this is seen as a discoloration of methylene blue). The recovered dye you can return the blue color, oxidizing oxygen in the air. Dehydrogenase milk at low temperature works slowly; after boiling milk it does loses activity. Hence, this enzyme also has an optimum reaction temperature.
In experiments with a slurry of muscle dehydrogenase succinic acid, like all enzymes lose activity after boiling. In the absence of succinic acid reaction is not (if you are well washed slurry with water and removed thus it contains succinic acid). The enzyme quickly oxidizes succinic acid to fumaric, transferring the hydrogen atoms on the dye.
And another experience with the same enzyme, but with a different object with microorganisms. Until now you had to deal with plant and animal tissues. However, microorganisms, perhaps even better armed enzymes because they have no other means of struggle for existence, in addition to the bio. With one of the microorganisms, more specifically, mushrooms, and we will deliver the experience. We are talking about ordinary Baker's yeast. Among the many active enzymes, which are produced by the yeast, there's a familiar dehydrogenase.
A piece of compressed yeast spread on a plate with two Channa tablespoons of boiled water. Grind better than plastic or aluminum spoon. When the mixture becomes homogeneous, make her clean dropper in two test tubes. They should pre-wash with soap and a cotton swab on a stick, washed with water and dried. Another pipette, add in both test tubes a little oil.
The tube 1 place for five minutes in a pot of boiling water and cool to room temperature. In both test tubes, add a pinch of sugar and gently stir so that the sugar is dissolved. The third pipette type in both test tubes for 10-15 drops of dye solution. Watch the colour of the solution.
The experience can be somewhat difficult, it will take more tubes. Try to change the temperature of the solution, to take different amounts of yeast and sugar. And conclusions provided you do yourself, without any leading questions. Now, when you have accumulated sufficient experience, you are quite a force.