4. Specificity of amylase and sucrase enzymes action.

The polysaccharides, such as starch and glycogen are specific substrates for amylase. The sucrose disaccharide serves as a substrate for the sucrase enzyme. The hydrolysis process with enzymes being present can be found in Trommer’s test that is not given by the initial carbohydrates.

They enumerate four test tubes. 2 ml of starch solution are poured into test tubes number 1 and 2; and 2 ml of sucrose solution are poured into test tubes number 3 and 4. 0,5 ml. of saliva solution are added to test tubes number 1 and 3, and 0,5 ml. of 1% yeast sucrase solution are added to test tubes number 2 and 4. Everything is mixed and placed in the water bath at a temperature of 38-40°С for 10 minutes. After cooling they test reactions with iodine to find starch in tests number 1 and 2 and then use Trommer’s test to find glucose in tests number 3 and 4. The final step is making a conclusion about the specificity of the learned enzymes.

sucrose glucose glycoside hydroxiles fructose

Test questions

1. What is the role of enzymes in an organism?

2. What class of chemical compounds do enzymes belong to?

3. What class do the enzymes used in the laboratory work belong to?

4. Why does inactivation of the enzymes solutions take place while boiling them?

5. How can you explain the specificity of sucrase and amylase enzymes? How can it be proved?

6. How does the lowering of pH influence the activity of α-amylase? Why?

7. On what principle is the qualitative definition of enzymes based on?

Laboratory work 5. Identifying the activity of enzymes

1. The influence of activators and inhibitors upon activity of enzymes.

1. The influence of activators and inhibitors upon α-amylase.

Pour 0,5 ml. of a 1% solution of sodium chloride into the first test tube, 0,5 ml of a 1% solution of copper sulfate into the second test tube, and 0,5 ml of distilled water into the third one. Add 1 ml of a 0,5% of starch solution and one drop of saliva dissolved with water (1:5) to each test tube. Mix the contents of the test tubes by shaking and place them into the water thermostat with the temperature of 37°С. In intervals of 1-2 minutes make tests from the test tubes and check the reaction with iodine solution in potassium iodide. The hydrolysis process reaches the stage of erythrodextrine. Mark the time (in minutes) of erythrodextrine appearance. Make a conclusion about the action (activator, inhibitor) of the investigated components.

1.2. Inhibitory effect of chloride ions on dehydrogenase complex of potatoes.

Potatoes cut into slices. One slice left to control, the second is sprinkled with sodium chloride, the third - potassium iodide, the fourth - potassium chlorate. After 15 - 20 minutes three slices become dark, and sprinkled with sodium chloride mains unchanged.

2. Identifying the activity of α-amylase according to Wolgemut.

The method is based on identifying the ultimate dissolving of α–amylase solution which still allows splitting of the given quantity of starch up to erythrodextrine under certain conditions. Wolgemut’s method can be used to approximately identify the activity of α–amylase in pancreatic juice, blood, urine and other biological liquids.

Put 1 ml of saliva into a test tube; add 9 ml of distilled water and mix. You get saliva solution 1:10.

Pour 1 ml of water into 10 enumerated test tubes. Add 1 ml of 10 times dissolved saliva to the first test tube by three times breathing in and out the liquid from the pipette. Then replace 1 ml of the liquid from the first test tube into the second one, mix the contents as it is mentioned before. Replace 1 ml of the liquid from the second test tube into the third one and so on.

Remove 1 ml of the liquid from the tenth test tube after mixing it.

Add 2 ml of 0,1% starch solution to all the test tubes beginning with the tenth one and mix the contents.

Place the test tubes into the thermostat at a temperature of 37°С for 30 minutes.

In 30 minutes cool the test tubes and add 1 drop of iodine solution in potassium iodide to all the test tubes. Mark the test tube where splitting of starch up to erythrodextrine has taken place and the latter gives red-brown coloring in reaction with iodine. The activity of α-amylase is expressed by the quantity of milliliters of 0,1% starch solution which can be split by 1 ml of undiluted saliva at a temperature of 37°С up to the stage of erythrodextrine during 30 minutes.

For example: if you observe red-brown coloring in the fourth test tube where the saliva is 160 times dissolved, it means that 1 ml of undiluted saliva would split 160 times more starch solution. Therefore, 1 ml of undiluted saliva splits during 30 minutes at 37°С: 2160 = 320 ml of 0,1% starch solution. Symbolically these are 320 units of α-amylase according to Wolgemut:

А 37°/30' = 320 un.