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enzymes explained

THE EFFECT OF VARIOUS FACTORS ON ENZYME ACTION

The enzyme you will use will be amylase, from a fungal source. Alternatively, salivary amylase from saliva, prepared amylase from the pancreatic juice, or "diastase" from plants could be used.

As with the previous work: The substrate will be starch. In these experiments, enzyme activity will be measured by the time taken for starch to disappear, monitored by testing with iodine/potassium iodide solution, using individual drops in a recessed tile. In other words, you will not be not testing for the products of the reaction, which you have already found out about.

This work will extend the theme of the previous work, by which you should have learned what amylase does to starch.
Each of the 4 experiments is designed to show the effect of a different ([continuously] variable) factor on the action of this enzyme on its substrate. In each case the principles behind the experiment will be similar, i.e. holding several factors constant whilst varying only one other.

Materials

1.5% amylase (+ plastic pipettes) - already in waterbaths

1% starch sol

iodine/potassium iodide (I/KI) solution - for testing

Expt 3 only: acid - 0.1M hydrochloric acid (HCl) alkali - 0.1M sodium carbonate

universal pH indicator and narrow range pH papers

Apparatus

5 boiling tubes (+ rack) 10ml measuring cylinder - for starch

plastic pipettes recessed tile paper towel marker pen timer

electrically heated waterbaths (36 C) - shared with next bench

Expt 2 only: also 0 C (ice-water), 18 C (Lab?) 36 , 54 , 72 C (electrically heated waterbaths)

Procedure

1) Each team will take 5 tubes, and number them 1-5.
According to the appropriate table overleaf, place the correct contents into each tube EXCEPT THE ENZYME AMYLASE.
2) Swirl the tube to mix the contents and place the tube into the appropriate waterbath, as indicated, and leave it for about 5 minutes to reach the appropriate temperature.
3) Place single drops of iodine solution in the recesses in the tile.
4) In each waterbath, there is a tube containing the amylase, and a plastic dropping pipette. This is for group use, so be sure not to contaminate it. Make sure you know what the divisions represent!
5) Synchronise the next actions amongst the team, and note the time:
One after another, add the amylase to each of your tubes of starch. Use a new plastic pipette to thoroughly mix the contents (leave the pipette in the tube), and leave the tube containing the mixture in the water bath.
6) Immediately, then at (3 minute?) intervals afterwards, use the pipette to remove a small amount of liquid from each tube and apply 2 drops of it to the iodine in the tile recess. Immediately record the result in the table (iodine either turns bluish black or stays reddish orange), then return the pipette to its tube and move on to the next tube in sequence. Take care not to cross-contaminate tubes, or suck back iodine. In the table note whether starch is still present (++, +) or absent (-). Intermediate (doubtful?) results may be obtained. The testing interval may be lengthened for some tubes after some time.
Stop testing when no starch is left in most tubes.

Experiment 3 only: use indicator paper to measure the pH of a drop of liquid from each tube, taken in the same way as the drops for testing on the tile. Record these results in the table on the last page.

Variations in experimental details (treatments)

Experiment 1

Tube
number
Temperature
/ C
Starch
/ml
Other
additions
Amylase
/ml
ADD LAST!
1 36 10 - 0.5
2 36 10 - 1.0
3 36 10 - 2.0
4 36 10 - 4.0
5 36 10 - 8.0

Experiment 2

Tube
number
Temperature
/ C
Starch
/ml
Other
additions
Amylase
/ml
ADD LAST!
1 0 10 - 2.0
2 18 10 - 2.0
3 36 10 - 2.0
4 54 10 - 2.0
5 72 10 - 2.0

Experiment 3

Tube
number
Temperature
/ C
Starch
/ml
Other
additions
Amylase
/ml
ADD LAST!
1 36 10 2.0 ml acid 2.0
2 36 10 0.5 ml acid 2.0
3 36 10 - 2.0
4 36 10 0.5 ml alkali 2.0
5 36 10 1.0 ml alkali 2.0

Experiment 4

Tube
number
Temperature
/ C
Starch
/ml
Other
additions
Amylase
/ml
ADD LAST!
1 36 2.5 - 2.0
2 36 5 - 2.0
3 36 10 - 2.0
4 36 20 - 2.0
5 36 40 - 2.0

Results table

specimen data?

Your experiment no. >

Reminder: Record dark or light blue-black as starch present, orangey-red as starch absent.

Time /min Presence (++,+) or absence (-) of starch in tube number
1 2 3 4 5
................. ................. ................. ................. ................. .................
................. ................. ................. ................. ................. .................
................. ................. ................. ................. ................. .................
................. ................. ................. ................. ................. .................
................. ................. ................. ................. ................. .................
................. ................. ................. ................. ................. .................
................. ................. ................. ................. ................. .................

When the starch is used up (result of the iodine test consistently turns from + to -) , the time taken for the change in your experiment can be transferred to the table below. If there is no such change, then the result should be recorded as "greater than" the last time of testing.(> x min )

Time taken for disappearance of starch in tube number
1 2 3 4 5
................. ................. ................. ................. .................

The whole class results will then be shared and transferred to tables overleaf, so that conclusions can be drawn from these by all class members, because these results form an integrated set of data.

Examine the tables detailing the treatments in each experiment in turn. You should be able to see the trend shown in the variations in the experimental details.

Which tube number has the same values for each of the factors in all of the experiments?

> 3

What are the exact values for these factors in this treatment?

> 2.0 ml amylase > temperature 36 C > no pH additions > 10ml starch


In the following section, remember that the shorter the time taken for starch to disappear, the higher the rate of reaction. Compare the results obtained in the various tubes. At each end of the table, put a simple description to explain how the treatments varied. Make sure you understand what you are asked.

What factor was varied in experiment 1? Which ones were held constant?

varied:>amount of amylase constant:>temperature> pH >starch concn

Give a title for experiment 1.

> The effect of amylase concn on the rate of starch disappearance

Summary of results:

treatment this end Time taken for disappearance of starch in tube number treatment this end
1 2 3 4 5
low enzyme ................. ................. ................. ................. ................. high enzyme

What do you conclude from these results?

>More amylase causes quicker breakdown of starch

What factor was varied in experiment 2? Which ones were held constant?

varied:>temperature constant:>amount of amylase >pH >starch concn

Give a title for experiment 2.

>The effect of temperature on the rate of starch disappearance

Summary of results:

treatment this end Time taken for disappearance of starch in tube number treatment this end
1 2 3 4 5
low temperature ................. ................. ................. ................. ................. high temperature

What do you conclude from these results?

> Amylase breaks down starch quicker at 54 or 36 and not at all at 0 or 72C.

What factor was varied in experiment 3? Which ones were held constant?

varied:> pH constant:> amount of amylase >temperature >starch concn

Give a title for experiment 3.

>The effect of pH on the rate of starch disappearance

Summary of results:

treatment this end Time taken for disappearance of starch in tube number treatment this end
1 - pH .. 2 - pH .. 3 - pH .. 4 - pH .. 5 - pH ..
low pH ................. ................. ................. ................. ................. high pH

What do you conclude from these results?

> Amylase works best in slightly acidic conditions.

What factor was varied in experiment 4? Which ones were held constant?

varied:>starch concn constant:> amount of amylase >temperature > pH

Give a title for experiment 4.

> The effect of quantity of starch on the action of amylase.

Summary of results:

treatment this end Time taken for disappearance of starch in tube number treatment this end
1 2 3 4 5
low substrate ................. ................. ................. ................. ................. high substrate

What do you conclude from these results?

>Smaller amounts of starch are broken down the quickest!



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