Experiments on Enzymes and Enzyme Action

In this article, we propose to discuss various experiments on enzymes and enzyme action.

A. Hydrolytic Enzymes:

I. Amylase (or Diastase):

(a) Make thin starch paste by shaking up a little ordinary starch or wheat flour with 50 ml boiling water and standing it to cool.

Get some germinating pea seedlings, 2-3 g. (in which the radicle has grown out at least an inch); remove the seed coats, grind the cotyledons with water in a mortar and filter through a funnel.

Pour the starch paste into three white saucers and label these. Leave saucer 1 as it is; into 2 pour a few drops of iodine soln. (it turns blue) and into 3 pour some of the watery extract from the cotyledons. Set the three saucers in a fairly warm place (about 35-40°C.) and from time to time test 1 and 3 with iodine soln.

Note that 3 soon gives only a reddish- brown colour with iodine soln. and ultimately remains uncoloured with iodine, showing that starch has disappeared—starch has been converted into sugar by the enzyme amylase which has been extracted from the cotyledons.

(b) Squeeze the milky contents of about half-a-dozen sprouting wheat grains into a test-tube with a little water and stir well. Filter and add a few drops of mixture of Fehling A and B to the clear filtrate and heat. A brick-red ppt. of Cu₂O indicates the presence of simple reducing sugars produced by the enzyme diastase in the germinating seeds.

II. Invertase:

Mix about 10 ml of yeast extract (1 g.) with 10 ml of a 2% soln. of pure sucrose (test it before to see whether it is non-reducing with Fehling soln.). Put the flask or beaker at about 38°C. After 30 mins. to 1 hr. add Fehling soln.

Observe considerable reduction of Fehling due to the conversion of sucrose into reducing glucose and fruc­tose. This may also be done with extracts of germinating seeds.

III. Lipases: Fat- or oil-splitting Enzymes or Esterases:

(a) Take some linseed oil (olive oil will also do) and test for acidity, if any Neu­tralise, if necessary. Take some germinating Ricinus seeds which have just begun to germinate, and crush or chop them up.

Add them to the oil mixture, shaking vigorously. Test again with litmus paper after a few hrs. The reaction is now markedly acidic due to the hydrolysis of fats into fatty acids and glycerol.

(b) (i) Take a small portion of the liquid in a test tube. Then add a little CuS0₄ soln. and a few drops of NaOH soln. A deep blue colouration is produced, but there is no ppt. of Cu(OH)₂ which is kept in solution by glycerol.

(ii) To another portion of the liquid add a little solid KHS0₄ and heat strongly; a pungent odour (acrylic aldehyde) confirms the formation of glycerol.

IV. Proteolytic Enzymes:

Trypsin:

Pound up some fresh tissue or seeds of peas and beans, carrot and cab­bage with 100 ml water containing a little toluene as preservative. Filter and then add a small quantity of peptone and shake up thoroughly. Keep in a warm place (about 35^oC.) for 2-3 days, then test for the amino acid, tryptophan. Filter the extract and then- acidify it with drops of acetic acid (dil).

Add bromide water carefully, drop by drop, until a purple colour develops, indicating the presence of tryptophan. Then shake up with a little amyl alcohol; on standing the alcohol will rise to the top carrying with it any of the purple colour formed.

B. Oxidation-Reduction Enzymes:

V. Direct Oxidases:

Cells containing oxidase systems become discoloured after injury due to the presence of these enzymes. The browning or discolouration of many tissues (e.g., pota­toes) and fruits when cut (as in apples, pears, etc.) is due to the activity of these oxidases.

Apply a drop or two of an alcoholic solution (freshly made) of gum guiacum (inner portion of a lump of gum may also do) on the cut open surface of a potato tuber. Dry the surface gently with a filter paper.

A deep-blue oxidation product (guiacum blue) is formed. Guiacum soln. is one of the most sensitive reagents used for oxidation as even the minutest trace of oxygen may be detected by this reagent.

VI. Indirect Oxidases or Peroxidases:

Cells of many plants cannot directly bring about aerobic oxidation of gum guia­cum. The peroxidases cannot turn guiacum blue until hydrogen peroxide is added.

Cells containing these enzymes do not turn brown when injured but if both H₂O₂ and gum guiacum are applied to the cut surfaces of horse-radish root, deep-blue colour is obtained.

Pyragallol, catechol and quinone may also be used instead of guiacum— (with pyragallol, the coloured product is red).

It has been shown that the presence of peroxidases in plant tissues may intensify the colour changes brought about by oxidases, when H₂O₂ is added.

VII. Dehydrogenases:

These enzymes bring about simultaneous oxidation-reduction reactions when, one compound is oxidised by removal of hydrogen and the other reduced (acceptor of hydrogen).

(a) In a test tube take 1 ml of a 15% solution of glucose and 1 ml of either living yeast cells or Baker’s yeast (zymin). Fill up the tube with a very dilute solution of methylene blue. Cork carefully and excluding all contact with external air place the tube for a short time in a warm place.

The dye methylene blue is seen to deco­lourise owing to the formation of leucomethylene blue, i.e., methylene blue is reduced by hydrogen derived from hydrogen donor, glucose, which is oxidised.

(b) If the tube containing colourless leucomethylene blue is uncorked and contents shaken in air, the blue colour reappears. This is because leucomethylene blue is auto- oxidisable; it can act as a hydrogen donor, giving up hydrogen readily to atmospheric oxygen which acts as a hydrogen acceptor, forming water.

VIII. Catalase:

Catalase is usually present in plant tissues but it does not bring about oxidation as in the case of other oxidation-reduction enzymes. The oxygen released by catalase is in the active form. Catalase prevents accumulation of H₂O₂ or organic peroxides in plant tissues. It is one of the most active enzymes that have yet been studied.

It is present in yeast cells (or in Zymase extract) and is thermolabile:

(a) In a test tube, which is half-filled with a mixture of equal volumes of H₂O₂ and water, add a few slices of freshly cut potato tuber. Evolution of oxygen bubbles can .be seen immediately.

(b) As in previous expt., add about 1 gm. of potato tuber (ground up with alcohol and filtered dry with a filter pump) in a test tube containing equal volumes (10 ml) of a mixture of water and H₂O₂.

Invert the open mouth of the tube (holding it by thumb) below the surface of water in a basin and hold it vertically by means of clamp arrangement. A rapid evolution of oxygen takes place which accumulates by displace­ment of solution in the tube.

(c) If the experiment is performed with vigorously boiled potato tuber tissue, no evolution of oxygen is seen, as heating destroys the enzyme.

(d) A suspension of Baker’s yeast (0.5—1.0 g.) added to a dilute solution of H₂O₂, liberates oxygen almost instantaneously.