Method 2. A method of measuring the reduction of substrate concentration
in an enzyme reaction
The starch
to maltose reaction - method 2 - monitoring a colour change
In this case you are detecting when the substrate (starch)
is used up when it is broken down to maltose by the enzyme amylase.
Enzyme reaction equation: starch +
water === amylase ==> maltose
2(C6H10O5)n
+ nH2O ====> nC12H22O11
Starch is a natural polymer where n is a very
large number.
This reaction is called a 'hydrolysis' because
a molecule reacts with water to give two or more products.
You can use 'baby rice' for this experiment!
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
The method for this particular reaction relies on
detecting the presence of starch using iodine (illustrated above).
You use a dilute solution of
iodine dissolved in potassium iodide solution, which is orangey-brown in
colour.
When the iodine solution is added to a starch solution a blue-black colour is seen.
This is
a simple standard biological laboratory test for starch and you simply take
a sample from the reaction mixture and test it for starch using the iodine
solution in the wells of a spotting tile.
When you no longer see a
blue-black colour, all the starch is used up and you note the time taken.
The rate of the enzyme reaction can be expressed
as the reciprocal of the reaction time:
1 / time in s-1
or min-1
This involves using a continuous sampling system -
all the experimental details further down.
Using a
colorimeter to follow the speed of a reaction
This method is fine in a school/college laboratory
where simple apparatus will be available for several groups in a class.
However it is possible to measure the intensity of the blue-black colour
using an electronic instrument called a colorimeter.
A sample of the
reaction mixture is diluted and put into a special tube and the colorimeter apparatus
can measure the intensity of the blue-black colour.
The dilution must done with the same volumes of
reaction mixture and water used to dilute it.
A special light
filter allows you to measure just the intensity of the colour you are
interested in.
You also take a blank reading without the starch present. From a
calibration graph of known starch concentrations versus their colour
intensity with iodine you can actually measure the concentration of the starch as it
decreases with time.
This method does avoid the uncertainty of when the
blue-black colour actually completely disappears. In the methods described
below you are measuring the average rate and the rate is varying all the
time. This colorimeter method avoids errors due to the varying speed of the
reaction, which is always slowing down!
Light from a suitable
source is passed through a light filter to select the most
appropriate wavelength of light, some of which is then absorbed by the
solution held in a special glass cuvet (a sort of 'test tube'). The amount of light
absorbed is called, and measured as, the absorbance which is a
function of the coloured solute concentration.
(a) The effect
of changing concentration for an enzyme reaction - method 2.
(starch to maltose
using the enzyme amylase)
(vary either the starch or amylase concentration)
Enzyme reaction equation: starch +
water === amylase ==> maltose
2(C6H10O5)n
+ nH2O ====> nC12H22O11
The theory of the method is explained at the start of
the method 2. section
The basic
experimental
procedure for method 2.
You can start with 1% starch solution and 1%
amylase solutions are suitable concentrations for this investigation.
The presence of starch is detected with
orangey-brown dilute iodine solution which gives a blue-black colour
with starch.
When all the starch is broken down, the iodine
solution colour is unchanged i.e. it remains an orangey-brown.
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).
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.
The boiling tubes of stock solutions of starch and
amylase of varying concentrations should be put into the water bath, so
everything involved is at the same start temperature.
A portion of dilute iodine solution is placed into
all the wells of the white spotting tile with a pipette.
Portions of the starch and amylase are carefully
measured out into a boiling tube and shaken well and immediately 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.
You need
x cm3 of amylase solution
(constant)
y cm3 starch solution
z cm3 water
x + y + z = constant volume.
y + z must be kept constant, but you can vary
them to give different concentrations of the starch.
The pH should stay constant, but if you think
you need one then you need a buffer solution that matches the
optimum pH of catalase. The volume of the buffer would have to be
kept constant.
Start the stopwatch. Once the experiment is
underway, with a clean teat pipette you sample every 10 seconds
and spot some of the reaction mixture into a well of the iodine
solution. At first the iodine solution is turned blue-black showing the
presence of starch.
Eventually the iodine solution is unchanged, no
blue-black colour observed, then you
note the total time elapsed.
Repeat several times so you can calculate a
more accurate average reaction time for the final analysis.
You then repeat the whole exercise with different
concentrations of starch, keeping the amylase concentration constant OR
varying concentrations of amylase and keeping the starch concentration
constant.
The reciprocal of the time (1 ÷ time) for the
starch to be used up gives you a relative measure of the rate of the enzyme
reaction.
(The rate has arbitrary units of reciprocal
seconds or minutes)
The rate of the enzyme reaction can be expressed
as the reciprocal of the reaction time:
rate proportional to 1 / time in s-1
or min-1
As the concentration of either starch or enzyme is
increased, the reaction time should decrease - upper graph on the right.
You then
draw a graph of the mean values of the rate of reaction (1/time) at each temperature versus concentration
- lower graph on the right. The graph should show an optimum value of
the maximum rate of reaction for that particular mixture.
See also GCSE chemistry notes:
Effect on rate of changing reactant
concentration in a solution
(b) The effect
of changing temperature
for an enzyme reaction - method 2.
(starch to maltose
using the enzyme amylase)
Enzyme reaction equation: starch +
water === amylase ==> maltose
2(C6H10O5)n
+ nH2O ====> nC12H22O11
The theory of the method is explained at the start of
the method 2. section
1% starch solution and 1% amylase solutions are
suitable concentrations for this investigation.
The presence of starch is detected with
orangey-brown dilute iodine solution which gives a blue-black colour
with starch.
When all the starch is broken down, the iodine
solution colour is unchanged i.e. it remains an orangey-brown.
The thermostated water bath should be set to an
initial temperature e.g. 20oC an the experiment repeated at
the same temperature raising the temperature to higher values e.g.
ideally the range from 25oC, 30oC, 35oC,
40oC, 45oC and 50oC.
The boiling tubes of stock solutions of starch and
amylase should be put into the water bath, so everything involved is at
the same start temperature.
A portion of dilute iodine solution is placed into
all the wells of the white spotting tile.
The basic
experimental
procedure for method 2.
Portions of the starch and amylase are carefully
measured out into a boiling tube which is immediately placed in the
water bath and start the stop watch. Make sure the boiling tube is fully
immersed in water so it and the contents are at the right temperature.
With a teat pipette you sample every 10 seconds
and spot some of the reaction mixture into a well of the iodine
solution. At first the iodine solution is turned blue-black showing the
presence of starch.
Eventually the iodine solution is unchanged, no
blue-black colour observed, then you
note the total time elapsed.
Repeat several times so you can calculate a
more accurate mean reaction time for the final analysis.
You then repeat the whole exercise at 30oC,
35oC, 40oC, 45oC and 50oC etc. adjusting the thermostat
temperature control.
The reciprocal of the time (1/time) for the
starch to be used up gives you a measure of the rate of the enzyme
reaction.
The rate of the enzyme reaction can be expressed
as the reciprocal of the reaction time:
1 / time in s-1
or min-1
e.g. if all the starch is gone in 90 seconds,
the rate = 1/90 = 0.011 (arbitrary units of reciprocal seconds)
You should find from 20oC to 40oC
a decrease in total reaction time for the starch to be used up, but an
increasingly longer reaction time from 50oC to 70oC.
You then draw a graph of the mean values of the
rate of reaction (1/time) at each temperature versus temperature.
The graph should show an optimum value of the maximum rate of reaction
for that particular mixture.
See also GCSE chemistry notes:
Effect on
rate of
changing the temperature of reactants
(c) The effect
of changing pH
for an enzyme reaction - method 2
(starch to maltose
using the enzyme amylase)
Enzyme reaction equation: starch +
water === amylase ==> maltose
2(C6H10O5)n
+ nH2O ====> nC12H22O11
The theory of the method is explained at the start of
the method 2. section
1% starch solution and 1% amylase solutions are
suitable concentrations for this investigation.
You need a good range of buffer solutions,
preferably at least five ranging 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 presence of starch is detected with
orangey-brown dilute iodine solution which gives a blue-black colour
with starch.
When all the starch is broken down, the iodine
solution colour is unchanged i.e. it remains an orangey-brown.
The basic
experimental
procedure for method 2.
The thermostated water bath should be set to a
suitable temperature e.g. 25oC to 35oC.
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.
The boiling tubes of stock solutions of starch,
amylase and various buffer solutions of differing pH should be put into
the water bath, so everything involved is at the same start temperature.
You can use plastic syringes to measure out
the volumes.
You should use different syringes for the
buffer solutions, starch solution and the amylase solution.
You
should keep the total volume of the final mixture constant, but varying
the buffer pH.
A portion of dilute iodine solution is placed into
all the wells of the white spotting tile.
Portions of the starch, amylase and buffer
solution are carefully measured out into a boiling tube which is
immediately placed in the water bath and start the stop watch.
Make sure the boiling tube is fully immersed in water so it and the
contents are at the right temperature.
One the experiment is underway, with a teat pipette you sample every 10 seconds
and spot some of the reaction mixture into a well of the iodine
solution. At first the iodine solution is turned blue-black showing the
presence of starch.
Eventually the iodine solution is unchanged, no
blue-black colour observed, then you
note the total time elapsed.
The rate of the enzyme reaction can be expressed
as the reciprocal of the reaction time:
1 / time
in s-1
or min-1
Repeat several times so you can calculate a
more accurate average reaction time for the final analysis.
You then repeat the whole exercise with buffers of
differing pH.
The reciprocal of the time (1/time) for the
starch to be used up gives you a measure of the rate of the enzyme
reaction.
(The rate has arbitrary units of reciprocal
seconds)
You then draw a graph of the mean values of the
rate of reaction (1/time) at each temperature versus the pH of the
buffer solution.
You should get a peak around pH 5, a bit like the
graph on the right.
See a
'decay' investigation using milk and lipase gcse biology
revision notes
Its part of the web page
Carbon cycle,
nitrogen cycle, water cycle, decomposition - decay investigation
Key points
Based on
the syllabus-specifications for students taking the AQA, Edexcel and OCR
GCSE level biology examinations (~US grades 9-10).
Key
ideas on apparatus, method and processing of results
Aim of experiment
To investigate how
amylase breaks down starch into maltose and how
changing temperature, pH, and
concentration affects this reaction, using iodine solution
as an indicator.
Investigating the
enzyme-catalysed hydrolysis of starch to maltose using
amylase and an indicator method. This covers apparatus, method, and
how to test the effects of temperature, pH, and enzyme concentration.
Background
Amylase is a biological
catalyst (enzyme) that hydrolyses starch (a polysaccharide) into maltose
(a disaccharide).
The presence of starch can
be detected with iodine solution: starch turns iodine blue-black,
while its disappearance (i.e. full hydrolysis) means the solution
remains brown/orange.
Apparatus and
Chemicals Required
-
Test tubes
-
Test tube rack
-
Stopwatch
-
Water bath (or beaker with
hot water)
-
Thermometer
-
Pipettes/droppers
-
Iodine solution
-
Spotting tile (dimple
tile)
-
Starch solution
-
Amylase solution
-
pH buffer solutions (e.g.
pH 4, 7, 9)
Experimental
Procedure
-
Set up
a water bath at your desired temperature (e.g. 20°C, 30°C, 40°C, etc.).
-
Place a labelled test
tube with 5 cm³ of starch solution and another with 5 cm³ of
amylase solution into the bath. Leave for 5 minutes to equilibrate.
-
Add 1 cm³ of
buffer solution to the starch if testing pH effects.
-
Prepare a spotting
tile with a drop of iodine in each dimple.
-
Mix the starch and amylase
solutions together in a clean test tube and start the stopwatch
immediately.
-
At 30-second
intervals, use a pipette to remove a drop of the reaction
mixture and add it to a drop of iodine on the tile.
-
Record the time
taken for the iodine to stop turning blue-black. This is the
endpoint—when starch has been fully digested.
-
Repeat for different
temperatures, pH levels, and
amylase concentrations.
Variables
Independent variables
(the ones you change):
-
Temperature
(e.g. 20°C to 60°C)
-
pH
(acidic, neutral, alkaline buffers)
-
Amylase
concentration (dilute to
varying levels)
Dependent variable
(what you measure):
Controlled variables
(what stays the same - for 'fair' test):
-
Volume and concentration
of starch
-
Volume of amylase (except
when testing concentration)
-
Volume of buffer
-
Timing intervals
-
Same source/type of enzyme
and starch
How Variables Affect
the Reaction
1.
Temperature
-
Low temperatures:
Molecules move slowly—reaction is slow.
-
Optimum
temperature (around 37°C):
Maximum enzyme activity.
-
High temperatures
(above 50°C–60°C): Enzyme denatures—active site changes
shape and becomes non-functional.
2.
pH
3.
Enzyme Concentration
-
More enzyme molecules
increase the rate of reaction—more active sites available.
-
Eventually, substrate
becomes limiting—further increases in enzyme concentration have
no effect.
Summary of learning objectives and key words or phrases
Know about the experimental methods for investigating the enzyme catalysed
hydrolysis of starch to maltose with amylase by amylase 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 catalysed hydrolysis of starch to maltose with amylase, including
the apparatus and chemicals required.
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