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Enzymes: 7. Method 1. Investigating the enzyme catalysed decomposition of hydrogen peroxide by catalase - effects of changing temperature, pH and concentration, apparatus and chemicals requires, description of experimental procedure

Doc Brown's Biology exam study revision notes

There are various sections to work through, after 1 they can be read and studied in any order.

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Sub-index of biology notes on enzymes and digestion


The decomposition of hydrogen peroxide by the enzyme catalase

In this experiment you are measuring the rate at which oxygen is formed from the enzyme catalase decomposing hydrogen peroxide.

Here, the product oxygen gas, provides the means of following the rate of the reaction.

Enzyme reaction equation: hydrogen peroxide === catalase ===>  water  +  oxygen (gas)

2H2O2(aq)  ====>  2H2O(l)  +  O2(g)

For this reaction there are three experimental situations are fully described

(a) Changing concentration of substrate molecule or enzyme

(b) Changing the temperature of the reaction mixture - looking for the optimum

(c) Changing the pH of the reaction mixture - looking for the optimum

(a) The basic procedures for method 1.

Method 1. A method of measuring the rate of product formation from an enzyme reaction

(a) Investigating the effect of changing the concentration for an enzyme reaction

(decomposition of hydrogen peroxide to water and oxygen using the enzyme catalase)

(vary either the hydrogen peroxide or the enzyme catalase).

Enzyme reaction equation: hydrogen peroxide === catalase ===>  water  +  oxygen (gas)

2H2O2(aq)  ====>  2H2O(l)  +  O2(g)

Mashed up potato made into a fine slurry diluted with acts as a source of the enzyme catalase. It needs to be well shaken before use so that each portion measured out has the same amount of catalase in it.

A slurry is a pulverized solid mixed in a liquid.

You need a series of hydrogen peroxide solutions of known different concentrations and a fixed concentration of the potato mix to investigate the effect of changing the hydrogen peroxide concentration.

You may have to do some 'trial and error' experiments to find out which amounts give 'reasonable' results.

You can also keep the hydrogen peroxide concentration constant and do the investigation with a set of different concentrations of the potato-catalase mixture.

The water bath is set to a constant temperature e.g. 25oC. The apparatus is setup as illustrated above.

The optimum conditions for human catalase are pH 7 (so no need for buffer) and 37oC

If no thermostated water bath is available you can get reasonable results if the laboratory temperature stays reasonably constant - but record and monitor the room temperature.

You can use a beaker of heated water but its difficult to keep it at a constant temperature.

The 'stock' solutions of potato-catalase or hydrogen peroxide should be initially in separate boiling tubes and placed in the water bath so that everything starts at the right temperature. Or, if no water bath available, they can be just put together in test tube racks on the laboratory bench, but you should monitor and record the room temperature.

Depending on what accuracy you require, you can measure out a fixed amount of the potato slurry and a varied amount of the same hydrogen peroxide solution into the boiling tube using a pipette, or 10 cm3 measuring cylinder or plastic syringe. You should keep the total volume of the reaction mixture the same.

There shouldn't be a need for a buffer, but the mixture should have a constant pH of ~7.

If in doubt build a fixed volume of a pH 7 buffer into your method.

You should make up the reaction mixture of hydrogen peroxide and potato slurry as quickly as possible and shake well.

Your reaction mixture to vary the hydrogen peroxide concentration may be as follows

w cm3 of buffer (if used)

x cm3 of potato slurry - kept constant

y cm3 of hydrogen peroxide solution - variable

z cm3 of water - variable

w + x + y + z = total constant volume and volume y + z must also be kept constant.

You can vary y and z to give different concentrations if different stock solutions of

By varying volumes y and z you can produce a range of hydrogen peroxide concentrations if a variety of stock solutions are not available..

The boiling tube and mixture is quickly connected to the delivery tube rubber bung and placed in the water bath and the stop watch started. Make sure the boiling tube is fully immersed in water so it and the contents are at the right temperature.

Start the stopwatch. You can now measure how much oxygen is formed in a set time e.g. 1 minute, and repeat the experiment several times with the same volumes of reactants at the same temperature.

This will allow a more accurate mean value of the rate of reaction to be used in the final analysis.

(Or set of volume readings for one run over a longer time, and plot graph of volume versus time and measure the initial gradient, but more work for repeats - see graph on the right)

However you get the results, the rate is calculated as follows:

From the initial gradient of the graph, the rate of enzyme reaction is expressed as:

rate = volume of O2 formed time taken (cm3/s)

(c) doc bYou then draw a graph of the mean values of the rate of reaction (in cm3/s) at each temperature versus concentration.

You should find that the rate increases with increase in either hydrogen peroxide or enzyme concentration, if you have been very accurate you may get a nice linear graph like the one on the right or else!

You then repeat the whole exercise with different concentrations of the enzyme using the kind of x + y + z 'recipe' described above using a fixed concentration of hydrogen peroxide.


Gas syringe system

It is possible to get more accurate results using a gas syringe system, as long as the flask can be set up in a water bath (omitted from the diagram below!) or the laboratory temperature stays constant.

The investigation is conducted in the same way as already described above.

Factors affecting the rates of Reaction - theory and methods of measuring the speed of a reaction (c) Doc Brown

You can get more accurate data of the volume of oxygen formed over time and from the graphs work out the initial rate of reaction from the initial gradient (see right-hand side of above diagram).

Graph line A (steeper gradient) compared to graph line B may represent (i) an increase in concentration of either substrate or enzyme, or (ii) an increase in temperature or (iii) a solution pH nearer the optimum value for that particular enzyme.

See also GCSE chemistry notes: Effect on rate of changing reactant concentration in a solution


(b) Investigating the effect of changing temperature for an enzyme reaction

(decomposition of hydrogen peroxide to water and oxygen using the enzyme catalase)

Enzyme reaction equation: hydrogen peroxide === catalase ===>  water  +  oxygen (gas)

2H2O2(aq)  ====>  2H2O(l)  +  O2(g)

Mashed up potato made into a fine slurry diluted with water acts as a source of the enzyme catalase. It needs to be well shaken before use so that each portion measured out has the same amount of catalase in it.

You need a hydrogen peroxide solution of known and constant concentration.

You may have to do some 'trial and error' experiments to find out which amounts give 'reasonable' results.

You should also use the same volume of the well shaken potato slurry.

The water bath is set to the start temperature e.g. 20oC. The apparatus setup is illustrated above.

You could start as low as 10oC perhaps by cooling the water with ice, not sure how well it would work?

The two 'stock' solutions should be initially in separate boiling tubes and placed in the water bath so that everything starts at the right temperature  - solutions and boiling tube.

Depending on what accuracy you require, you can measure out a fixed amount of the potato slurry and a fixed amount of the same hydrogen peroxide solution into the boiling tube using a pipette or a 10 cm3 measuring cylinder or plastic syringe. You should keep the total volume of reaction mixture constant.

(There shouldn't be a need for a buffer, the mixture should have a constant pH of ~7)

(If in doubt use a buffer to match the optimum pH of the enzyme catalase).

You make up the reaction mixtures as quickly as possible in a boiling tube and shake well.

The boiling tube and mixture is quickly connected to the delivery tube rubber bung and the stop watch started.  Make sure the boiling tube is fully immersed in water so it and the contents are at the right temperature.

Start the stopwatch. You can now measure how much oxygen is formed in a set time e.g. 1 minute, and repeat the experiment several times with the same volumes of reactants at the same temperature.

Repeats will allow a more accurate average value of the rate of reaction to be used in the final analysis.

  (or set of volume readings for one run, plot graph of volume versus time and measure the initial gradient, but more work for repeats - see the graph on the right)

From the initial gradient of the graph, the rate of enzyme reaction is expressed as:

rate = volume of O2 formed/time taken (cm3/s)

(c) doc bYou then repeat the whole exercise at 30oC, 40oC, 50oC etc. adjusting the thermostat temperature control.

You should find from 20oC to 40oC an increase in the rate of oxygen production, but an increasingly slower rate of reaction from 50oC to 70oC (see graph on bottom right).

You then draw a graph of the mean values of the rate of reaction (in cm3/s) at each temperature versus temperature.

See the end of method 1. (a) for a gas syringe method

See also GCSE chemistry notes: Effect on rate of changing the temperature of reactants


(c) Investigating the effect of changing pH for an enzyme reaction

(decomposition of hydrogen peroxide to water and oxygen using the enzyme catalase)

Enzyme reaction equation: hydrogen peroxide === catalase ===>  water  +  oxygen (gas)

2H2O2(aq)  ====>  2H2O(l)  +  O2(g)

Mashed up potato made into a fine slurry diluted with water acts as a source of the enzyme catalase. It needs to be well shaken before use so that each portion measured out has the same amount of catalase in it.

You need a hydrogen peroxide solution of known and constant concentration AND stock solution of the potato slurry to provide the enzyme catalase.

You may have to do some 'trial and error' experiments to find out which amounts give 'reasonable' results.

You need a range of at least five stock solutions of buffers giving a variety of pH values e.g. ideally from pH 2 to pH 11.

A buffer solution keeps the pH constant in a reaction medium - it can neutralise small amounts of acid or alkali formed.

The water bath is set to a constant temperature e.g. 25oC-35oC.

The higher temperature is faster - do a trial run, if too slow raise the temperature, but don't go above 35oC).

The apparatus setup is illustrated above.

If no thermostated water bath is available you can get reasonable results if the laboratory temperature stays reasonably constant - measure and monitor.

The 'stock' solutions of catalase, hydrogen peroxide and the buffer solutions should be initially in separate boiling tubes and placed in the water bath so that everything starts at the right temperature. Or, if no water bath available, they can be just together in test tube racks on the laboratory bench, but you should monitor and record the room temperature.

Depending on what accuracy you require, you can measure out a fixed amounts of the potato slurry, hydrogen peroxide solution and the buffer solutions into the boiling tube using a pipette or more accurately with a 10 cm3 measuring cylinder.

Whatever your 'recipe', keep the total volume of the three solutions constant for the final reaction mixture.

The three solutions are mixed in a boiling tube and well mixed, the total volume should be constant and you use the same concentrations of the hydrogen peroxide and potato-catalase. The pH of the buffer should be the only variable.

The boiling tube and mixture is quickly connected to the delivery tube rubber bung and the stop watch started.  Make sure the boiling tube is fully immersed in water so it and the contents are at the right temperature.

Start the stopwatch. You can now measure how much oxygen is formed in a set time e.g. 1 minute, and repeat the experiment several times with the same volumes of reactants at the same temperature.

This will allow a more accurate mean value of the rate of reaction to be used in the final analysis.

(or set of volume readings for one run, plot graph of volume versus time and measure the initial gradient, but more work doing repeats - see the graph on the right)

From the initial gradient of the graph, the rate of enzyme reaction is expressed as:

rate = volume of O2 formed/time taken (cm3/s)

(c) doc bYou then repeat the whole exercise with different pH buffer solutions.

You then draw a graph of the mean values of the rate of reaction (in cm3/s) versus the pH, and it 'may' look like the graph on the right.

See the end of method 1. (a) for a gas syringe method


Summary of learning objectives and key words or phrases

Know about the experimental methods for investigating the enzyme catalysed decomposition of hydrogen peroxide by catalase and how to investigate the effects of changing temperature, changing pH and changing the concentration.

Know what apparatus is needed and the main points of the experimental procedure for the enzyme decomposition of hydrogen peroxide, including the apparatus and chemicals required.


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