Practical 3

Practical 3: Experimental investigation of a factor affecting enzyme activity. (Topic 2: Molecular Biology- 2.5 Enzymes).

The emphasis of this practical should be on designing experiments which control all important variables. You should be able to calculate rate of enzyme activity and produce and interpret graphs to show the expected effects of temperature, pH and substrate concentration on the activity of enzymes. You should be able to explain the patterns or trends apparent in these graphs.

Below you can download a sheet with some possible starting points for designing and investigation on enzymes.

How to calculate rate from a graph (such as one produced on logger pro).

Or in other words: change/time

In the experiment below, the oxygen produced at different temperatures was measured using an oxygen pressure sensor. The image below shows an example of the graph produced by logger pro at a given temperature:

By looking at the steepest part of the gradient (the initial reaction) the rate can be calculated:

This results of the experiment are below:

Graph of processed data:

Practice trying to describe and explain the results above. What were the strengths of this investigation? How could this investigation have been improved?

If the time taken for a reaction to happen is the outcome being measured as the Dependent Variable then calculate the rate by using the following equation:

1/t where t= time taken

You need to be able to interpret the graphs produced and compare them to expected data.

Effect of Temperature:

Effect of pH:

Effect of Substrate Concentration:

Here is an example of a protocol that could be adapted for an investigation to look at factor affecting the rate of enzyme activity:

Using the Information below design an experiment to investigate the effect of a variable on the activity of Lipase.

Basic Protocol:

c Label a test tube with the temperature to be investigated.

d Add 5 drops of phenolphthalein to the test tube.

e Measure out 5 cm³ of milk using a measuring cylinder (or syringe) and add this to the test tube.

f Measure out 7 cm³ of sodium carbonate solution using another measuring cylinder (or syringe) and add this to the test tube. The solution should be pink.

g Place a thermometer in the test tube. Take care as the equipment could topple over.

h Place the test tube in a water bath and leave until the contents reach the same temperature as the water bath.

i Remove the thermometer from the test tube and replace it with a glass rod.

j Measure out 1 cm³ of lipase from the beaker in the water bath for the temperature you are investigating.

k Add the lipase to the test tube and start the stopclock/ stopwatch.

l Stir the contents of the test tube until the solution loses its pink colour.

m Stop the clock/ watch and note the time in a suitable table of results.

Health and Safety

Sodium carbonate solution, 0.05 M. Make with 5.2 g of anhydrous solid, or 14.2 g of washing soda per litre of water. See CLEAPSS Hazcard; it is an IRRITANT at concentrations over 1.8 M.

Ethanol (IDA) in the phenolphthalein indicator is described as HIGHLY FLAMMABLE on the CLEAPSS Hazcard (flash point 13 °C) and HARMFUL (because of presence of methanol).

Glassware is breakable.

Electric water baths should be safety checked in accordance with your employer’s instructions.

Take care with thermometers and brief students how to react if they are broken.