Core practical 1 Teacher sheet

EDEXCEL Biology B Teacher Resource Pack 1 Investigate a factor affecting the initial rate of reaction

Core practical 1: Investigate a factor affecting the initial rate of

reaction

Objectives
● To be able to measure the initial rate of enzyme activity
● To understand why measuring the initial rate is important
Safety Specification links
● Trypsin solution at concentrations of 1% or above ● Practical techniques 1, 2, 3, 6 (and
is an irritant. It may cause an allergic reaction in 12 if a datalogger is used)
those allergic to products such as washing ● CPAC 1a, 2a, 2b, 3a–3c, 4a, 4b, 5a
powder.
● Wash splashes from the skin as quickly as
possible.
● Wear eye protection.
Procedure Notes on procedure
Milk protein (casein) is broken down by protease ● A check should be done before the
enzymes such as trypsin. The opaque white colour of lesson to ensure that the milk powder
the milk is replaced by a clear solution. Light passes suspension provides an absorbance
more easily through the final solution, so the reaction value within a suitable range when
can be monitored using a colorimeter or light sensor. first mixed with the enzyme. Further
1. Plan how you will dilute the 1% trypsin stock dilution of the milk may be required,
solution with distilled water to produce additional depending on the brand used. Note
test solutions of 0.2%, 0.4%, 0.6% and 0.8%. Aim that absorbance does not have true
3
to produce 10 cm of each concentration. Once units, although changes in
checked, make up the solutions as planned. ‘absorbance units’ may be discussed.
2.
3
Place 2 cm of trypsin solution and 2 cm of
3 ● Watch that students do not
distilled water into a cuvette. Use this as a contaminate the stock milk
reference cuvette to set the colorimeter suspension with trypsin as they are
absorbance to zero. transferring solutions to the cuvettes.
3.
3
Measure 2 cm of milk suspension into a second ● To get repeat measurements when
cuvette. colorimeters are limited, each group
3 can make up one set of
4. Add 2 cm of trypsin solution to the milk in the concentrations and then pool class
cuvette. Working quickly, mix and place the results before calculating mean
solution into the colorimeter and start the stop values to use in the absorbance/time
clock. graphs.
5. Measure absorbance immediately and then at 15 ● Inexpensive simple colorimeters can
second intervals (or more frequently if recording be constructed using a light-emitting
electronically) for 5 minutes, or until there is little diode (LED), a light-dependent
change in absorbance. resistor (LDR), a suitable resistor and
6. Rinse the cuvette with distilled water and repeat an ammeter. Various designs can be
for each concentration. found using an Internet search.

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 Core practical 1 Teacher sheet
EDEXCEL Biology B Teacher Resource Pack 1 Investigate a factor affecting the initial rate of reaction

Answers to questions
−1
1. Independent: trypsin concentration. Dependent: rate of reaction in absorbance units, s .
2. Because the reaction is rapid and the milk (substrate) concentration quickly declines. The rate
slows as the substrate is used up. Comparisons can only be made at the start of the reaction
where controlled variables such as substrate concentration are the same for all levels of the
independent variable.
3. A systematic error, because it would cause absorbance readings to be higher than the true
value for every measurement.
4. pH – the rate of reaction of enzymes varies with pH, due to changes in the shape of the active
site. An enzyme would have the highest rate of reaction at its optimum pH. A buffer might be
used to maintain pH at a suitable level.
Temperature – the rate of reaction of enzymes varies with temperature. As temperature
increases, particles gain more energy and more collisions take place between enzyme and
substrate particles. Enzymes have an optimum temperature at which the rate of reaction is at
its peak. Above that temperature, enzymes will begin to denature, changing the shape of the
active site and preventing further catalysis. A water bath and thermometer could be used to
maintain a suitable temperature.
Sample data

Absorbance/absorbance units

Trypsin concentration (%) 0 s 15 s 30 s 45 s 60 s 75 s 90 s
1.0 1.97 1.38 0.68 0.26 0.19 0.16 0.13

0.8 1.97 1.39 0.75 0.23 0.13 0.11 0.09

0.6 1.97 1.42 0.87 0.41 0.14 0.10 0.06

0.4 1.97 1.57 1.21 0.84 0.47 0.30 0.11

0.2 1.97 1.78 1.51 1.28 1.02 0.82 0.58

0.0 1.80 1.80 1.80 1.80 1.80 1.80 1.80

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need to adapt it to local circumstances. This document may have been altered from the original 2
 Core practical 1 Student sheet
EDEXCEL Biology B Teacher Resource Pack 1 Investigate a factor affecting the initial rate of reaction

Core practical 1: Investigate a factor affecting the initial rate of

reaction

Objectives
● To be able to measure the initial rate of enzyme activity
● To understand why measuring the initial rate is important
Safety All the maths you need
● Trypsin solution at ● Recognise and make use of appropriate units in
concentrations of 1% or calculations.
above is an irritant. It may ● Use an appropriate number of significant figures.
cause an allergic reaction in
those allergic to products ● Construct and interpret frequency tables and diagrams, bar
such as washing powder. charts and histograms.

● Wash splashes of trypsin ● Translate information between graphical, numerical and

from the skin as quickly as algebraic forms.
possible. ● Plot two variables from experimental or other data.
● Wear eye protection. ● Calculate rate of change from a graph showing a linear
● Inform the teacher if any relationship.
trypsin gets into your eyes. ● Draw and use the slope of a tangent to a curve as a
measure of rate of change.
Equipment
● skimmed milk powder suspension (2%)
● standard protease (trypsin) solution (1%) IRRITANT
● 6 test tubes and holder
● stop clock
3
● two 5 cm pipettes (or syringes/measuring cylinders)
● eye protection
● access to colorimeter (see diagram) (or light meter with datalogger)
● 2 cuvettes
● distilled water
Diagram

fig A A simple colorimeter.

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Users may need to adapt the risk assessment information to local circumstances. This document may have been altered from the original 1
 Core practical 1 Student sheet
EDEXCEL Biology B Teacher Resource Pack 1 Investigate a factor affecting the initial rate of reaction

Procedure
Milk protein (casein) is broken down by protease enzymes such as trypsin. The opaque white colour
of the milk is replaced by a clear solution. Light passes more easily through the final solution and so
the reaction can be monitored using a colorimeter (see diagram) or light sensor.
1. Plan how you will dilute the 1% trypsin stock solution with distilled water to produce additional
3
test solutions of 0.2%, 0.4%, 0.6% and 0.8%. Aim to produce 10 cm of each concentration.
Once checked, make up the solutions as planned.
3 3
2. Place 2 cm of trypsin solution and 2 cm of distilled water into a cuvette. Use this as a
reference cuvette to set the colorimeter absorbance to zero.
3
3. Measure 2 cm of milk suspension into a second cuvette.
3
4. Add 2 cm of trypsin solution to the milk in the cuvette. Working quickly, mix and place the
solution into the colorimeter and start the stop clock.
5. Measure absorbance immediately and then at 15 second intervals (or more frequently if
recording electronically) for 5 minutes, or until there is little change in absorbance.
6. Rinse the cuvette with distilled water and repeat for each concentration.
Analysis of results
1. Record your results in a suitable table.
2. Plot a graph of absorbance against time. It should be possible to plot each concentration as a
different line on the same axes.
3. Use the graph to determine the initial rate of reaction for each concentration. Do this by drawing
a tangent to the initial part of each curve and calculating the gradient of each line.
4. Draw a second graph to show the initial rate of reaction against the concentration of the
enzyme.
5. Write a short conclusion to describe and explain the result of this investigation.
Learning tips
● Use a sharp pencil when drawing graphs. Using different symbols around plotted points will
help to distinguish lines when several concentrations are plotted on to one set of axes.
Remember to include a key.
● Keep graph scales simple. Using one large square to represent 5, 10 or 20 (or perhaps 0.05,
0.1 or 0.2) is ideal when plotting intermediate points, as the smaller squares will have values
that are easy to work with.
Questions
1. What were the independent and dependent variables in this investigation?
2. Why is it important to measure the initial rate of the reaction rather than an average rate over a
longer time period?
3. If the surface of the cuvette is scratched, it can result in a greater absorbance of light. If the
cuvette used for the reaction was scratched (but the reference cuvette was not), would this give
a random or a systematic error? Explain your answer.
4. Suggest two variables that would normally be controlled in enzyme-catalysed reactions but
which have not been specifically controlled in this investigation. Explain why they would usually
be carefully controlled and suggest how this could be done.

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Users may need to adapt the risk assessment information to local circumstances. This document may have been altered from the original 2
 Core practical 1 Technician sheet
EDEXCEL Biology B Teacher Resource Pack 1 Investigate a factor affecting the initial rate of reaction

Core practical 1: Investigate a factor affecting the initial rate of

reaction

Objectives
● To be able to measure the initial rate of enzyme activity
● To understand why measuring the initial rate is important
Safety
● Enzyme powders and concentrated solutions are irritants. Refer to CLEAPSS Hazcard 33
(enzymes).
● They may produce allergic reactions and can be sensitisers (causing allergic reaction on
subsequent exposure). This is particularly common in those allergic to products such as
washing powder.
● They can cause asthma and can irritate the eyes, nose and skin.
● Avoid skin contact and inhalation.
● Wear disposable gloves and eye protection.
● Use a fume cupboard when handling enzyme powders.
● Wipe up solution spills or any traces of powders with a damp cloth.
● Rinse with plenty of water in case of contact with skin.
● If eyes are contaminated, irrigate for at least 10 minutes and see a doctor.
● Seek medical help if inhalation causes breathing difficulties.
Equipment per student/group Notes on equipment
3
skimmed milk powder suspension Make up with 2 g of skimmed milk powder in 100 cm water.
(2%) High fat content milk powders do not give good results.
3
Each student or group will need 5 cm for every concentration
3
tested (30 cm in total if one repeat of each concentration is
carried out).

3
standard protease (trypsin) solution Mix 1 g trypsin powder in 100 cm water. Add enough alkali
(1%) IRRITANT (e.g. dilute sodium hydroxide) while mixing it up to produce a
pH of 9. When making up the enzyme solution do not heat to
a temperature greater than 40 °C to dissolve.
Students will dilute this standard solution to give 0.2%, 0.4%,
3
0.6% and 0.8%. To make up 10 cm of each concentration
3
every student or group needs a total of 30 cm .
6 test tubes and holder
stop clock
3
two 5 cm pipettes (or
syringes/measuring cylinders)
eye protection
access to colorimeter Use the colorimeter with a red filter. Colorimeter output
(or light meter with datalogger) should be sent to a datalogger or computer if possible.
CLEAPSS has developed an inexpensive colorimeter that
uses paired LEDs. See the CLEAPSS website for details.
2 cuvettes
distilled water

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Users may need to adapt the risk assessment information to local circumstances. This document may have been altered from the original 1
 Core practical 1 Technician sheet
EDEXCEL Biology B Teacher Resource Pack 1 Investigate a factor affecting the initial rate of reaction

Notes

Practical activities have been safety checked but not trialled by CLEAPSS. © Pearson Education Ltd 2015
Users may need to adapt the risk assessment information to local circumstances. This document may have been altered from the original 2