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enzymes explained

- The action of lipase on fats

The substrate used in this experiment is fat, which makes up about 3.9% of full cream milk. A small amount of alkali has been added in order to change its pH and for experimental reasons.
The enzyme lipase is from the mammalian digestive system. Depending on the conditions in the surrounding medium, it may break down (digest) the fat to fatty acids and glycerol.
During this practical session you will be seeing the lipolytic effect of the enzyme (as well as the contribution made by the emulsifying effects of bile salts).
The fatty acids which are among the products of the reaction above will react with alkali to cause a change in pH, which will make the indicator phenolphthalein change from pink to colourless.

Preparation of a series of different combinations

Make sure that pipettes are not contaminated by placing them in other liquids.

- Do not use pipettes for any other purpose.

1) Place 6 test-tubes in a rack, and label them 1-6.

2) Using a single 3ml pipette (twice) put 6 ml of alkaline milk into tubes 1-6.

3) Using a single (smaller) pipette, add 0.5 ml phenolphthalein into tubes 1 to 5 BUT NOT 6.

4) Using a single different pipette, add 1 ml bile salts into tubes 1 , 2, 3 and 6 ONLY.

5) Using yet another pipette, add 1 ml lipase into tubes 1, 2, 4 and 6 ONLY.

6) Record the colours of the contents of the tubes in the table below - in the row labelled "colour at start".

7) Transfer tubes 2-6 to a water bath at 37 °C, and leave them for some time. In the meanwhile, you have the opportunity to carry out the test procedure overleaf which will tell you more about these substances.

8) At regular intervals examine the tubes and look for signs of colour changing from pink to whitish. Record the final colours of the contents of the tubes in the table below - in the row labelled "colour at end ".

Tube number 1 2 3 4 5 6

Contents Quantity (ml)
alkaline milk 6 6 6 6 6 6
phenolphthalein 0.5 0.5 0.5 0.5 0.5 -
bile salts 1 1 1 - - 1
lipase 1 1 - 1 - 1
Temperature/ °C lab 37 37 37 37 37
RESULTS colour at start pinkish pinkish pinkish pinkish pinkish whitish
colour at end whitish whitish pinkish whitish pinkish whitish
click for photographs

Which of the "contents "above is not normally found in the human small intestine?

> phenolphthalein

What is meant by the term lipolytic ?

> breaking down fats (lipids)into simpler substances

What is meant by the term emulsifying ?

> breaking (fat) droplets into smaller ones

Why do you think that the milk was made alkaline before the experiment? (2 reasons)

> because lipase acts in the alkaline conditions of the intestine

> when the fatty acids are produced, they neutralise the alkali and cause the indicator to change colour

Whereabouts in the body is lipase produced, i.e. Which organ makes it?

> pancreas

Whereabouts in the body is lipase released, i.e Where does it mix with "food" to be digested?

> duodenum

What sort of physical conditions exist there?

>alkaline (warm, wet, etc)

Whereabouts in the body is bile produced, i.e. Which organ makes it?

> liver

Whereabouts in the body is bile stored?

> gall bladder

What do you think is the role of bile salts in this experiment?

> to emulsify fat, i.e. make droplets smaller and increase the surface area so as to make it more easily broken down by lipase

Which tube or tubes shows/show a change first?

> 2 ?

What combination of ingredients and conditions do they include?

> lipase, bile salts, warm

Compare the result from this tube with the others. Write your conclusions from this experiment. This should include answers to questions such as:

What does lipase do to the fat in milk? What other factors are needed?Are they absolutely necessary?

> In order to break down fats into fatty acids, lipase needs warmth etc, and bile salts speed it up, but are not essential.


Whilst you are waiting for results from the above procedure, you should perform the biuret test (separately) on the spare lipase and milk [and the (1%) amylase from other experiments] that you have been using:

What is the biuret test used to identify?

> protein

Pour about 10 ml of sodium (or potassium) hydroxide into a boiling tube.

CARE! Wear eye protection when handling alkalies.

Add about 1 ml of copper sulphate solution; the colour should change to a deeper blue. Mix carefully. This is biuret reagent.

Place about 2.5 ml of each substance to be tested into a tube in the rack.

To each add about 2.5 ml of the blue mixture (biuret reagent) produced above.

Wait for a different colour to develop, compared with the leftover untouched mixture.

Test substance Resulting colour Conclusion : substances present
lipase purple/lilac protein
milk mauve protein
amylase purple/lilac protein

click for photographs What is your general conclusion from these tests?

>The enzymes lipase and amylase are/contain protein - also milk is a protein

Finally: Pour away the contents of ALL the tubes down the sink, then rinse the tubes with a gentle flow of cold water before placing them in the washing up bowl.

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