Worksheet EXPERIMENT 3.2: THE
THERMODYNAMIC PARAMETERS OF THE
THERMAL DENATURATION OF TRYPSIN
18 November 2020
13:51
Aims of the · To determine the activation energy for an enzyme
experiment catalysed reaction
· To determine whether the thermal denaturation of
trypsin is entropy or enthalpy driven
Your learning · To interpret data obtained by spectrophotometry to
objectives determine the reaction rate of an enzyme
· To gain experience in calculating thermodynamic
parameters from reaction rate data
PREPARATION
Before you come to the session, you should read the background materials on
Minerva, read through the experimental protocol, and using the information from
these sources, complete the preparation MCQ questions. This session will need
you to use Excel to process the data. If you are not familiar with any of the graph
plotting features used in this session, please ask your group demonstrator or
check Hints for using Excel for graph plotting.
SESSION ORGANISATION
All timings are approximate.
10:00-10:15: Make sure you arrive in good time. Download a copy of the session
worksheet from Minerva before or as soon as you arrive.
10:15-10:30: Brief introduction by the academic lead together with any course-
related announcements.
10:30-12:45 (including 15 mins coffee/tea breaks 11:30-11:45): You will enter
the breakout room with your group. Complete worksheet in your groups with help
,from your demonstrator. Remember that while you are working as a group, each
of you must complete their own worksheet, in your own words.
12:45-13:00: Conclusion and questions by the academic lead.
INTRODUCTION
This experiment concerns the thermal denaturation of the proteolytic enzyme,
trypsin. Since the rate of an enzyme-catalysed reaction is proportional to the
enzyme concentration (when the substrate is in excess), we can use values of
reaction rates to measure the concentration of the active form of the enzyme.
It is convenient to use a substrate which gives a product which absorbs within the
operating range of a spectrophotometer. In this experiment, the artificial
(“chromogenic”) substrate N-benzoyl-D-L-arginine p-nitroanilide has been used.
The product formed when this molecule is hydrolysed by trypsin is yellow and
absorbs at 405 nm. The change in absorbance at 405 nm against time indicates
the reaction rate.
By measuring reaction rate at a range of temperatures, it is possible to calculate a
number of thermodynamic parameters for the reaction, including the activation
energy for the reaction, and DSo and DHo for the denaturation process. These
values allow us to determine whether the thermal denaturation process is driven
by entropy (DSo) or enthalpy (DHo).
From an Arrhenius plot of rates measured over a range of temperatures, we can
determine the activation energy (Ea) of the reaction, and because it allows us to
calculate what the reaction rates would have been if there had been no thermal
denaturation, it enables us to calculate the value of Keq at each temperature.
The way in which the position of equilibrium varies with temperature is indicated
by the equilibrium constant,
E (active) ↔ E (denatured) Keq = [Edenatured] / [Eactive]
, From the way Keq varies with temperature we can use the van ’t Hoff plot to
determine the thermodynamic parameters, DSo and DHo for the denaturation
process.
(a) Determination of the Activation energy (Ea) for the trypsin-catalysed
hydrolysis of proteins
The Method used to obtain this data is outlined below. By now, these
instructions should be familiar to you.
N.B. On this occasion you will not carry out the experiment yourself; use the data
provided to calculate activation energy.
Reagents: Phosphate buffer
Substrate stock (N-benzoyl-D-L-arginine p-nitroanilide)
Trypsin Solution
· Fill the cuvette with phosphate buffer (1.5 ml), distilled water (1.25 ml) and the
substrate stock solution (0.2 ml).
· Fill another cuvette with distilled water and use it to zero the
spectrophotometer at 405 nm.
· Place the cuvette in the waterbath and leave for 10 min to allow the solution to
reach a temperature of 28°C. Check temperatures with the thermometer.
· When the solutions have reached the required temperature, move the cuvette
to the spectrophotometer, add 0.05 ml of trypsin solution and stir, without
removing from the cell housing.
· Measure the absorbance at 405 nm every 15 seconds for 180 seconds (twelve
readings in all). Speed is of the essence to ensure accurate initial readings.
· Repeat for other temperatures
THERMODYNAMIC PARAMETERS OF THE
THERMAL DENATURATION OF TRYPSIN
18 November 2020
13:51
Aims of the · To determine the activation energy for an enzyme
experiment catalysed reaction
· To determine whether the thermal denaturation of
trypsin is entropy or enthalpy driven
Your learning · To interpret data obtained by spectrophotometry to
objectives determine the reaction rate of an enzyme
· To gain experience in calculating thermodynamic
parameters from reaction rate data
PREPARATION
Before you come to the session, you should read the background materials on
Minerva, read through the experimental protocol, and using the information from
these sources, complete the preparation MCQ questions. This session will need
you to use Excel to process the data. If you are not familiar with any of the graph
plotting features used in this session, please ask your group demonstrator or
check Hints for using Excel for graph plotting.
SESSION ORGANISATION
All timings are approximate.
10:00-10:15: Make sure you arrive in good time. Download a copy of the session
worksheet from Minerva before or as soon as you arrive.
10:15-10:30: Brief introduction by the academic lead together with any course-
related announcements.
10:30-12:45 (including 15 mins coffee/tea breaks 11:30-11:45): You will enter
the breakout room with your group. Complete worksheet in your groups with help
,from your demonstrator. Remember that while you are working as a group, each
of you must complete their own worksheet, in your own words.
12:45-13:00: Conclusion and questions by the academic lead.
INTRODUCTION
This experiment concerns the thermal denaturation of the proteolytic enzyme,
trypsin. Since the rate of an enzyme-catalysed reaction is proportional to the
enzyme concentration (when the substrate is in excess), we can use values of
reaction rates to measure the concentration of the active form of the enzyme.
It is convenient to use a substrate which gives a product which absorbs within the
operating range of a spectrophotometer. In this experiment, the artificial
(“chromogenic”) substrate N-benzoyl-D-L-arginine p-nitroanilide has been used.
The product formed when this molecule is hydrolysed by trypsin is yellow and
absorbs at 405 nm. The change in absorbance at 405 nm against time indicates
the reaction rate.
By measuring reaction rate at a range of temperatures, it is possible to calculate a
number of thermodynamic parameters for the reaction, including the activation
energy for the reaction, and DSo and DHo for the denaturation process. These
values allow us to determine whether the thermal denaturation process is driven
by entropy (DSo) or enthalpy (DHo).
From an Arrhenius plot of rates measured over a range of temperatures, we can
determine the activation energy (Ea) of the reaction, and because it allows us to
calculate what the reaction rates would have been if there had been no thermal
denaturation, it enables us to calculate the value of Keq at each temperature.
The way in which the position of equilibrium varies with temperature is indicated
by the equilibrium constant,
E (active) ↔ E (denatured) Keq = [Edenatured] / [Eactive]
, From the way Keq varies with temperature we can use the van ’t Hoff plot to
determine the thermodynamic parameters, DSo and DHo for the denaturation
process.
(a) Determination of the Activation energy (Ea) for the trypsin-catalysed
hydrolysis of proteins
The Method used to obtain this data is outlined below. By now, these
instructions should be familiar to you.
N.B. On this occasion you will not carry out the experiment yourself; use the data
provided to calculate activation energy.
Reagents: Phosphate buffer
Substrate stock (N-benzoyl-D-L-arginine p-nitroanilide)
Trypsin Solution
· Fill the cuvette with phosphate buffer (1.5 ml), distilled water (1.25 ml) and the
substrate stock solution (0.2 ml).
· Fill another cuvette with distilled water and use it to zero the
spectrophotometer at 405 nm.
· Place the cuvette in the waterbath and leave for 10 min to allow the solution to
reach a temperature of 28°C. Check temperatures with the thermometer.
· When the solutions have reached the required temperature, move the cuvette
to the spectrophotometer, add 0.05 ml of trypsin solution and stir, without
removing from the cell housing.
· Measure the absorbance at 405 nm every 15 seconds for 180 seconds (twelve
readings in all). Speed is of the essence to ensure accurate initial readings.
· Repeat for other temperatures
