The below mentioned article includes a collection of seven experiments on enzyme activity.
1. Experiment to demonstrate the activity of enzymes:
Benzidine solution, razor, thin sections of actively growing root (or germinating seeds or germinating pollen grains), phosphate buffer, hydrogen peroxide (1%), ammonium chloride (5%), starch paste.
Method, observations and results:
1. Cut thin sections of fast-growing roots or germinating seeds or take some pollen grains and put the material in phosphate buffer at pH 7.0. Now transfer the material in the incubation mixture [made of 5% benzidine saturated solution + 5 ml hydrogen peroxide (1%) and 1 ml ammonium chloride (5%)]. Incubate the entire mixture at room temperature for 5 minutes and observe. Dark-blue colour appears. This is due to the presence of the enzyme peroxidase.
2. Make a paste of starch, immerse in it the provided material like actively growing roots or germinating pollen grains and test for sugar. Positive test of sugar indicates that starch has transformed into sugars, and this further confirms the presence of enzyme amylase in the studied plant material.
2. Experiment to demonstrate that heat destroys activity of enzyme but not that of a catalyst:
Four test tubes, manganese dioxide (MnO2), water, beaker, spirit lamp, piece of fresh liver or potato, hydrogen peroxide, boiled and cooled piece of liver or potato, boiled and cooled manganese dioxide solution.
Pour 2 ml of H2O2 solution is each of the four test tubes. Add a pinch of manganesedioxde in the first test tube, pre-boiled and cooled 1 ml of manganese dioxide solution in the second, small piece of fresh liver or potato in third and pre-boiled and cooled piece of liver or potato in the fourth.
All the test tubes are kept at room temperature if it is summer and warm water (kept at about 38°C) if it is winter. Oxygen bubbles are found to come out of solution in the first three test tubes but not in the fourth one.
Manganese dioxide is a catalyst that helps to break hydrogen peroxide into water and oxygen. The catalyst is not affected by heat as it is clear from evolution of oxygen in the second test tube.
Fresh liver or potato contains enzymes peroxidase and catalase that help in evolving oxygen from hydrogen peroxide. The enzymes are functional at room temperature (test tube three) but heating destroys their activity (test tube four). Therefore, heat kills enzyme activity but has little effect on the activity of a catalyst.
(i) Test tubes must be thoroughly washed with water before use.
(ii) Do not perform the experiment without warm water if it is cold outside,
(iii) Keep the temperature of warm water to about 38″C. Do not allow it to go beyond 50°C.
(iv) Use fresh potato slice or liver piece.
3. Experiment to prove that enzymes are specific in their activity:
Four test tubes, one percent starch solution, one percent sucrose solution, saliva, Bennedict’s or Fehling’s solution, spirit lamp.
Take four test tubes. Pour 1 ml of 1% starch solution in the first, 1 ml of 1% sucrose solution in the second, 1 ml each of 1% starch solution and saliva in the third, and 1 ml each of 1% sucrose solution and saliva in the fourth. Keep all the test tubes undisturbed for one hour at room temperature if it is summer and in warm water (about 38°C) if winter.
Afterwards pour 3 ml of Bennedict’s or Fehling’s solution (having cupric ions) in each of the test tube. Heat the test tube to boiling for about 2 minutes and observe. There is no change of colour in first and second test tubes. The fourth test tube also does not show any change in colour. However, in the third test tube, the blue colour of Bennedict’s or Fehling’s solution gets changed to yellowish or reddish precipitate.
The change of colour from blue to yellowish or reddish precipitate is caused by conversion of cupric ions of Bennedict’s or Fehling’s solution into cuprous oxide.
This occurs in the presence of reducing sugars. Both sucrose and starch are non-reducing in nature as is found by the absence of colour change in test tube one and two. In test tube four having sucrose and saliva there is no enzyme activity since colour change is absent.
However, test tube three having starch and saliva (containing enzyme salivary amylase) reducing sugars are produced as is clear from change of colour. Therefore, the enzyme, salivary amylase, present in saliva catalyses hydrolysis of starch but not sucrose which is a common disaccharide.
(i) Test tubes must be thoroughly washed and dried;
(ii) The water in which the solutions are to be kept must not be very hot.
(iii) In winter warm water must be taken,
(iv) Before the collection of saliva, mouth must be washed thoroughly with distilled water,
(v) Swill the rinsed mouth with 7-10 cc of distilled water for 1 minute and then collect the same, as water containing saliva,
(vi) While heating the solution should not be allowed to bump violently.
4. Experiment to demonstrate the activity of the enzyme amylase extracted from the germinating barley or pea seeds:
Starch powder, iodine solution, germinating barley or pea seeds, distilled water, test tubes, mortar, pestle, filter paper, and funnel.
1. Take a little amount of ordinary starch and make a thin paste of it in about 50 ml boiling water. Allow it to cool down.
2. Take about 5 gm. of germinating seeds of barely or pea. In case of pea seedlings, remove their cotyledons. Ground the cotyledons along with distilled water in the mortar, and filter the contents through a funnel.
3. Pour the starch solution in two test tubes and mark them as ‘A’ and ‘B’.
4. Add a few drops of iodine solution in tube ‘A’ and observe the colour change.
5. Add the filterate of the crushed cotyledons or endosperm of barely in tube ‘B’.
6. Keep both the tubes at a warm place (about 35°- 40°C) for about 30 minutes.
In test tube ‘A’, the contents turn blue in colour. In test tube ‘B’, the contents show reddish- brown colour. After about 15 minutes, if iodine solution is added, it shows no positive test for starch in tube ‘B’. However, if a few drops of Fehling’s solution are added in tube ‘B’, a brick-red precipitate appears.
Formation of blue colour in tube ‘A’ confirms the test of starch. In tube ‘B’, formation of reddish-brown colour is due to the fact that addition of seed extract supplied the enzyme amylase which partially hydrolysed the starch into maltose (a 12-carbon sugar). After about 30 minutes, the entire starch in tube ‘B’ gets completely hydrolysed into hexose sugar.
Due to this the iodine solution gives negative test for starch. Formation of reddish-brown colour after the addition of Fehling’s solution confirms the presence of hexose sugar. It is a reducing sugar, and a product of hydrolysis of starch made of amylase and amylopectin.
The enzyme amylase is present in the germinating barley or pea seeds. It is released during the crushing process. Amylase is actually an enzyme which catalyzes the breakdown of starch into monosaccharide units.
5. Experiment to study the enzyme activity of diastase in germinating seeds of barley and to study the influence of pH and temperature:
The enzyme diastase acts on starch and converts it to hexose sugar.
Germinating barley seeds, mortar, water, muslin cloth, centrifuge, measuring flask, iodine solution prepared in potassium iodide), starch solution, test tubes, beaker, enzyme extract, pipette, Benedict’s solution, buffer solutions of known pH, diastase solution, water baths (7), stop watch.
Preparation of Required Solutions:
(a) Starch solution:
It is prepared by adding 2 gm. of soluble starch in 50 ml of boiling water.
(b) Buffer solution A:
Add 6.95 gm. of monobasic sodium phosphate (0.2 M) in 250 ml of distilled water and use it as buffer solution A.
(c) Buffer solution B:
Dissolve 17.92 mg of dibasic sodium phosphate (Na2HPO4.12H2O) in 250 ml of water to get 0.2 M buffer solution B.
(d) Iodine solution (1 %):
Mix 1 gm. iodine and 2 gm. KI in 300 ml water and use it as iodine solution.
Method and observations:
1. Take 10 gm. of germinating barley seeds and 20 ml of water and grind them in mortar.
2. Filter the above mixture through muslin cloth, centrifuge the filtrate at low speed, take the supernatant liquid in a measuring flask and make up the volume of the enzyme extract upto 100 ml.
3. Take six test tubes and put 1 ml iodine solution (1 %) in each of them. Also add 20 ml water in each of them.
4. In a separate test tube take 1 ml of 1% iodine solution and 20 ml water and add 1 ml starch solution. This will work as a control.
5. Take 10 ml of starch solution in a beaker, add 1ml enzyme extract and shake it well.
6. This starch diastase mixture is now called digestion mixture. Pipette out 1ml of this digestion mixture and add it in each of the six test tubes containing iodine solution and observe. Colour starts disappearing. Note the time of disappearance of colour.
7. After about 10 minutes of digestion, take out 1ml of digestion mixture into a test tube, and now test for sugar by Benedict’s reagent. Sugar test is positive.
This shows that activity of the enzyme diastase has transformed the starch into sugar. This further confirms the enzyme activity of diastase in germinating seeds of barley.
Effect of pH:
Take 9 test tubes and in each of them take 5ml of buffer solution of known pH like 5.0,5.5, 6.0,6.5,7.0,7.5,8.0,8.5,9.0. In each test tube, add 5 ml of starch solution (1%). Now add 1 ml of diastase solution in each of the test tubes and note the time of addition. Shake the contents thoroughly and mix them well.
Now pipette 1 ml of the reaction mixture every 5 minutes into separate test tubes containing 1ml of iodine solution and 20 ml of water. Take a graph paper and plot the time taken in minutes for complete hydrolysis (as shown by complete disappearance of colour of iodine) against the pH. Time of disappearance of colour is different in different pH, and this shows the influence of pH on enzyme activity.
Effect of temperature:
Take seven water baths and maintain them at seven different temperatures like 100°C, 80°C, 60°C, 40°C, 20°C, 10°C and 0°C. Take seven test tubes and in each of them add 5 ml of soluble starch (1 %) maintained at pH7 and put them in seven different water baths maintained at different temperatures.
Note the time when contents in different test tubes attain the temperature of their respective water baths and now add 1 ml of diastase solution in each test tube. Shake the test tubes well, wait for 5 minutes and add in each test tube 1 ml mixture of 1 ml iodine solution and 20 ml water. Wait for some time, note the time taken for disappearance of iodine colour against each temperature and plot the time on graph paper.
6. Experiment to demonstrate the activity of peroxidase in plant material:
Potato tuber, test tube, muslin cloth, hydrogen peroxide (3%), pyrogallol solution (1%).
1. Macerate 5 gm. of potato tuber, squeeze it through a muslin cloth, and take 3 ml of the potato extract in a test tube.
2. Put 1% solution of pyrogallol (a phenolic compound) in the test tube containing potato extracts and add 3 drops of hydrogen peroxide (3%) and observe.
Change in colour takes place.
This change in colour shows the presence of peroxidase activity in the extract of potato tuber.
7. Experiment to demonstrate the pH change inhibits in enzyme activity:
Two test tubes. 1% starch solution, saliva, dilute HCI, beaker, water, spirit lamp, iodine solution (I + KI).
Pour 2 ml of starch solution in each of the two test tubes. Add 1 ml of fresh saliva in each. A few drops of dilute hydrochloric acid are added to one of the test tubes to make its solution acidic. Both the test tubes are kept for one hour at room temperature if it is summer or in warm water (about 38°C) if it is winter.
After one hour both the test tubes are tested for starch by pouring 1 to 2 drops of iodine solution. Test tube one shows negative starch test while test tube two (acidified) develops blue colour showing the presence of starch.
In test tube one, pH is nearly that of saliva. The salivary amylase contained in saliva is functional and causes hydrolysis of starch because starch test is negative.
In test tube two appearance of blue colour indicates that the starch has not been hydrolysed by enzyme present in saliva. The only difference in two test tubes is that the solution of the second test tube has been acidified. Therefore, change in pH inhibits enzyme activity.
(i) Test tubes must be thoroughly washed and dried,
(ii) Before the collection of saliva mouth must be washed and any acidic or alkaline food must not be taken,
(iii) Warm water must be used in winter,
(iv) Care must be taken that there is no contamination of first test tube by the HCI which is being used for second test tube.