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Inside every cell, hundreds of chemical reactions are occurring simultaneously on the complex infrastructure of the cytoplasm.  An enzyme is a protein that speeds up one or more biological reactions.  In biological systems, reactions may occur very slowly, or not at all, in the absence of an enzyme.  Enzymes will greatly increase the rate of formation of the product.  Enzymes can increase the rate of reactions by a factor of at least a million.

Introduction

Hypothesis

The hypothesis that I am going to test during this investigation is:

Increasing the temperature increases the rate of an enzyme controlled reaction.

Background information

Inside every cell, hundreds of chemical reactions are occurring simultaneously on the complex infrastructure of the cytoplasm.  An enzyme is a protein that speeds up one or more biological reactions.  In biological systems, reactions may occur very slowly, or not at all, in the absence of an enzyme.  Enzymes will greatly increase the rate of formation of the product.  Enzymes can increase the rate of reactions by a factor of at least a million.
Unlike chemical catalysts, enzymes are specific.  This means that each enzyme will normally only catalyse one reaction.  The substance that the enzyme combines with is called the substrate, which combines with the enzyme at a particular place on the enzyme’s surface called the active site.
Enzyme molecules are usually much larger than their substrates, with the active site forming only a small part of the enzyme.  The rest of the enzyme’s structure is involved in maintaining the shape of the active site.
In living cells, most chemical reactions require an input of energy before the molecules will react together.  This is referred to as the activation energy.  Enzymes increase the rate of reactions by reducing the free energy of activation, so that the barrier to a reaction occurring is lower in the presence of an enzyme.  The combination of enzyme and substrate produces a new energy profile for the reaction, with a lower activation energy.
However, enzymes need very specific conditions to function properly.  They are very susceptible to changes in their environment, for example, variations in the pH of the liquid they are in can dramatically alter their performance.  They are also affected by temperature, as a temperature over about 40oC will break the bonds that hold together the active site, so the enzyme molecule will no longer fit the substrate molecule.  The Q10 law states that for every 10oC rise in temperature, the rate of reaction will double, up to about 40oC.

Key variables

To test out my hypothesis I am going to look at how temperature affects the catalysis by amylase of the breaking down of starch.  In this experiment the changing variable is temperature.  All of the other variables- enzyme and substrate concentration and the volume of reactants used will remain the same.

Predictions

In this experiment, I predict that as the temperature increases, so the rate of reaction will increase.  However, once the temperature passes 40oC, the rate will fall rapidly, as the enzyme is denatured by the heat.  To be more specific, I predict that for every 10oC rise in temperature, the rate of reaction will double, because of the Q10 law.


Method

Method

Apparatus

100cm3 1% starch solution
200cm3 1% amylase solution
3 spotting tiles
Iodine solution
Benedicts reagent
Test tubes
Ice
Bunsen burner
Tripod
Gauze mat
250cm3 beaker
Stopwatch
 0-100oC thermometer
 Pipette
 Matches

Procedure

Before the experiment was begun, samples of the starch and amylase solutions were tested with Benedicts reagent to show that no reducing sugars were present.  If they had been then the experiment would have proved nothing.
Once it had been established that no reducing sugars were present, three spotting tiles were prepared by having a drop of iodine placed into each cavity.  A waterbath was then set-up at 10oC and 5cm3 of starch and 10cm3 of amylase in test tubes were left to acclimatise for five minutes.  The amylase and starch were then mixed, the stopwatch started and a drop removed using a pipette and placed in a cavity.
Every thirty seconds a drop was removed and placed in a cavity on a spotting tile.  This was continued until the iodine no longer turned a blue/black colour.  After the experiment had continued for ten minutes the interval between samples was increased to a minute.  The time taken until the iodine no longer turns blue/black was recorded and the experiment repeated twice and the average result calculated.  The experiment was then repeated at temperatures of 20oC, 30oC, 40oC, 50oC, 60oC, 70oC, 85oC and 100oC.
The average time and rate of reaction at each temperature was calculated and graphs of time against temperature and rate against temperature were plotted.
A control experiment was also carried out, whereby the amylase was boiled so that the enzymes were denatured.  There was no result for this experiment.


Results

Results

The table below shows the results that I obtained from this experiment.  On the following two pages are graphs of temperature against time and temperature against rate (1/time).

Results Table

Graph showing time taken for reaction to take place at various temperatures

Graph showing rate of reactions at different temperatures

Graph showing rate against 1/temp


Discussion

Discussion and conclusions

What my results show

The Q10 law reads as follows:

For every 10oC rise in temperature, the rate of reaction will double, up to about 40oC, when the enzyme starts to be denatured.

As you can see from the tables and graphs of my results, my results roughly fit this pattern.  However, in my experiment, the rate of reaction continues to increase when the temperature rises above 40oC because the amylase that I used was `industrial` amylase; that is, it is designed to function in conditions that would normally result in the enzyme being denatured.
My results appear to fit the pattern suggested by the Q10 law reasonably well up until 40oC, as the rate of reaction roughly doubles each with each temperature rise (6.41x10-4 is approximately double 2.98x10-4, and 1.333x10-3 is approximately double 6.41x10-4 and so on).  After 40oC however, the rate of increase falls to less than double.  This is probably because some, but not all, of the enzymes are being denatured.  The continued rise in rate can be attributed to the fact that an increasing number of the collisions between enzyme molecules and substrate molecules take place with more energy than the activation energy of the reaction.  After 85oC, the enzyme behaved typically, as its activity dropped of very quickly, as `normal` enzymes do.  By 100oC there was no activity from the enzyme at all.
The graphs of time against temperature and rate against temperature both show how the speed of reaction increases as the temperature increases.  The time against temperature graph shows how the time taken for the reaction to finish decreases dramatically whilst the increase in temperature remains the same.  As I have said above, this is because more of the collisions between molecules happen with enough energy to make the reaction proceed.
The rate against temperature graph shows how the rate increases with temperature.  The graph has a fairly typical shape, except that there is no decrease in rate as the temperature rises.

Limitations in my experiment


Discussion Continued...

Anomalous results

If you look at the graph of rate against temperature you can see that the result for 70oC appears to be a little slower than it should be to fit in with the pattern shown on the rest of the graph.
The slower rate at 70oC was almost certainly caused by an experimental error, for example, the waterbath not being at 70oC or the starch and amylase not being given enough time to acclimatise to the new temperature.  However, all three results at 70oC are within fifteen seconds of each other, so it is more likely that one of the results around it is wrong.  As the result at 60oC fits in with the pattern shown by the rest of the graph, I think it is more likely that the result at 85oC is inaccurate.  Also, one of the results at 85oC is a little out from the others.  Although it is only twenty-five seconds, it is approximately 30% out from the other two.
As I have previously said, the results obtained for temperatures above 40oC do not really fit into the pattern suggested by the Q10 law, so in a sense they are also anomalous results.  This decrease in the increase in rate can be attributed to the fact that at the higher temperatures, some of the amylase is being denatured.  However, the rate of reaction continues to increase because of the energy with which the molecules collide.

Biological significance of my results

The fact that enzyme activity increases with temperature accounts for why homeostasis and thermoregulation is so important in living organisms.  Natural enzymes stop working at above 45oC, so it is important that organisms can maintain their body temperature at the temperature which will result in optimum enzyme performance and therefore the fastest metabolism.  All organisms are dependant on the hundreds of reactions that make up their metabolism and if the rate of metabolism falls then the organism may die.
Enzymes increase the rate of these reactions, so without them the reactions would proceed so slowly as to be unnoticeable.  It is therefore vital for living organisms to maintain optimum conditions for these enzymes.
The reaction that I studied, between starch and amylase to make glucose, is very important in the human body because if it did not happen then the starch that we eat would be indigestible.  This would result in a reduced supply of glucose, leading to a reduced supply of energy, because glucose is the main molecule involved in glycolysis and the Krebs Cycle to produce ATP.
It is important that the results I obtained are significant because if they were not then there would be no point to homeostasis and so the energy which the body uses maintaining the constant internal environment would be wasted.