Core Practical 4 - 08/06/2022
Core practical 4 - investigate the effect of enzyme and substrate concentrations on
the initial rates of reactions
Aim : To investigate how enzyme concentration can affect the initial rate of reaction
Introduction
Enzyme is a catalytic protein which can speed up the chemical reaction without being altered
in the reaction process. In the reaction process, there is no bond formed between the
enzyme and the substrate, so the enzyme goes back to its original shape and can be used
again (Creative Enzymes, 2022). There are 2 theories explaining the enzyme-substrate
interaction : in the lock and key model, the active site of an enzyme is precisely chapped to
hold specific substrate whereas in the induced fit model, the active site and substrate don't fit
perfectly but both alter their shape to connect (Castro, 2014).
In the digestive system, enzymes help the body break down larger complex molecules into
smaller molecules which can be absorbed. (Newman, 2018). For example, Trypsin is a
proteolytic enzyme which is formed in its inactive form ‘trypsinogen’ in the exocrine cells of
the pancreas cells and is secreted into the lumen of the intestine. The enzyme hydrolyzes
bonds in proteins into peptides and amino acids (Rick, 1974).
A colorimeter can be used to measure the change in opacity through the absorbance value.
Calorimeters measure absorbance of light waves by comparing the amount of light getting
through a solution with the amount that can get through a sample of pure solvent.
Calorimeters contain a photocell which is able to detect the amount of light passing through
the solution under investigation. The current produced by the photocell depends on the
quantity of light hitting it after passing through the coloured solution. A colorimeter takes
three wideband readings along the visible spectrum to obtain a rough estimate of a colour
sample (Choudhury, 2014).
Hypothesis
Increasing the concentration of enzymes directly increases the rate of reaction when
sufficient substrate is present. This is because there will be more active sites available for
free substrates to form enzyme-substrate complexes and therefore more products per unit
time are formed. Milk powder contains a white protein called casein. A white suspension of
milk clears on the addition of the enzyme trypsin (Laulainen, 2015). As the casein in milk
breaks down, the smaller molecules become soluble, thereby reducing the opacity of the
fluid (Southern Biological, 2022).
Therore, as time goes on, the concentration of trypsin increases, the transmittance value will
increase as the solution will be less opaque and more light will be transmitted.
Risk assessment
Hazard & risk Precaution
Protease enzyme solution - Most enzymes Wear gloves, lab coats and goggles to
are sensitisers and may cause allergy or minimise skin contamination. Any spillages
asthma or breathing difficulties if inhaled. onto the skin should be washed off
immediately. Enzyme adequate ventilation
, Core Practical 4 - 08/06/2022
in the room.
Trypsin is an irritant which can cause Wear gloves, lab coats and goggles to
irritations to the skin. minimise skin contamination. Any spillages
onto the skin should be washed off
immediately. Cover any open wounds.
Glasswares such as test tubes are fragile Take care when handling and keep away
causing them to break easily. They have from the edge of surfaces.
very sharp edges when they’re broken, this
may cause skin injury
Variables
Independent variable : concentration of protease solution ranging from 0% to 1% used
Dependent variable : the initial rate of reaction calculated from the rate at which the
transmittance value (au) measured decreases as the solution becomes less opaque.
Control variables
- Volume of substrate - 2cm3 of milk solution will be used each time to keep a
constant amount of E-S complexes that can form so only the effect of changing the
number of active sites can be measured.
- Concentration of substrate - same conc should be used for repeats as changing
the concentration would affect the amount of E-S complexes that can be formed
- Keep the temperature of the room the same by performing the practical in one go
in a short amount of time to prevent drastic room temperature changes - higher
temperatures increase the rate of reaction as the particles gain more energy and
more successful collisions occur. Lower temperatures slows the speed of the
particles so less E-S complexes form.
- Same amount of mixing of the reactant together as mixing more would increase the
number of collisions and therefore increase the rate of reaction.
- The same colorimeter should be used for all the solutions which are calibrated using
distilled water. This makes sure all the values are relative to the water and changing
colorimeter would need to be recalibrated each time and may give different results
- Do not touch the clear part of the cuvette as this will leave fingerprints.
Fingerprints absorb and scatter light slightly despite not being readily visible which
would affect the transmittance reading.
- Size of cuvette for colorimeter and the same type of the cuvette is the same used for
all trials since different types of cuvette are made with different materials which may
affect the experiment when using the colorimeter as they perform differently. The
cuvette was calibrated at the beginning.
Control
Enzyme concentration 0.0% (distilled water) can be used and added to the casein solution.
Water should not have any effect on the opacity of the casein solution so can be used to
compare the effect of the enzyme solution.
Equipment
Core practical 4 - investigate the effect of enzyme and substrate concentrations on
the initial rates of reactions
Aim : To investigate how enzyme concentration can affect the initial rate of reaction
Introduction
Enzyme is a catalytic protein which can speed up the chemical reaction without being altered
in the reaction process. In the reaction process, there is no bond formed between the
enzyme and the substrate, so the enzyme goes back to its original shape and can be used
again (Creative Enzymes, 2022). There are 2 theories explaining the enzyme-substrate
interaction : in the lock and key model, the active site of an enzyme is precisely chapped to
hold specific substrate whereas in the induced fit model, the active site and substrate don't fit
perfectly but both alter their shape to connect (Castro, 2014).
In the digestive system, enzymes help the body break down larger complex molecules into
smaller molecules which can be absorbed. (Newman, 2018). For example, Trypsin is a
proteolytic enzyme which is formed in its inactive form ‘trypsinogen’ in the exocrine cells of
the pancreas cells and is secreted into the lumen of the intestine. The enzyme hydrolyzes
bonds in proteins into peptides and amino acids (Rick, 1974).
A colorimeter can be used to measure the change in opacity through the absorbance value.
Calorimeters measure absorbance of light waves by comparing the amount of light getting
through a solution with the amount that can get through a sample of pure solvent.
Calorimeters contain a photocell which is able to detect the amount of light passing through
the solution under investigation. The current produced by the photocell depends on the
quantity of light hitting it after passing through the coloured solution. A colorimeter takes
three wideband readings along the visible spectrum to obtain a rough estimate of a colour
sample (Choudhury, 2014).
Hypothesis
Increasing the concentration of enzymes directly increases the rate of reaction when
sufficient substrate is present. This is because there will be more active sites available for
free substrates to form enzyme-substrate complexes and therefore more products per unit
time are formed. Milk powder contains a white protein called casein. A white suspension of
milk clears on the addition of the enzyme trypsin (Laulainen, 2015). As the casein in milk
breaks down, the smaller molecules become soluble, thereby reducing the opacity of the
fluid (Southern Biological, 2022).
Therore, as time goes on, the concentration of trypsin increases, the transmittance value will
increase as the solution will be less opaque and more light will be transmitted.
Risk assessment
Hazard & risk Precaution
Protease enzyme solution - Most enzymes Wear gloves, lab coats and goggles to
are sensitisers and may cause allergy or minimise skin contamination. Any spillages
asthma or breathing difficulties if inhaled. onto the skin should be washed off
immediately. Enzyme adequate ventilation
, Core Practical 4 - 08/06/2022
in the room.
Trypsin is an irritant which can cause Wear gloves, lab coats and goggles to
irritations to the skin. minimise skin contamination. Any spillages
onto the skin should be washed off
immediately. Cover any open wounds.
Glasswares such as test tubes are fragile Take care when handling and keep away
causing them to break easily. They have from the edge of surfaces.
very sharp edges when they’re broken, this
may cause skin injury
Variables
Independent variable : concentration of protease solution ranging from 0% to 1% used
Dependent variable : the initial rate of reaction calculated from the rate at which the
transmittance value (au) measured decreases as the solution becomes less opaque.
Control variables
- Volume of substrate - 2cm3 of milk solution will be used each time to keep a
constant amount of E-S complexes that can form so only the effect of changing the
number of active sites can be measured.
- Concentration of substrate - same conc should be used for repeats as changing
the concentration would affect the amount of E-S complexes that can be formed
- Keep the temperature of the room the same by performing the practical in one go
in a short amount of time to prevent drastic room temperature changes - higher
temperatures increase the rate of reaction as the particles gain more energy and
more successful collisions occur. Lower temperatures slows the speed of the
particles so less E-S complexes form.
- Same amount of mixing of the reactant together as mixing more would increase the
number of collisions and therefore increase the rate of reaction.
- The same colorimeter should be used for all the solutions which are calibrated using
distilled water. This makes sure all the values are relative to the water and changing
colorimeter would need to be recalibrated each time and may give different results
- Do not touch the clear part of the cuvette as this will leave fingerprints.
Fingerprints absorb and scatter light slightly despite not being readily visible which
would affect the transmittance reading.
- Size of cuvette for colorimeter and the same type of the cuvette is the same used for
all trials since different types of cuvette are made with different materials which may
affect the experiment when using the colorimeter as they perform differently. The
cuvette was calibrated at the beginning.
Control
Enzyme concentration 0.0% (distilled water) can be used and added to the casein solution.
Water should not have any effect on the opacity of the casein solution so can be used to
compare the effect of the enzyme solution.
Equipment
