Abigail Kirchner and Levi Hongsermeier

Dr. Moser
20 March 2016
Instructors Manual for Transfer Energy and the Hydrophobic Effect
Overview
This lab is aimed at teaching the students about the hydrophobic effects seen in
protein folding and the effect of cosolvents by way of experimenting with transfer
energy and absorbance. Students will measure absorbance of aqueous solutions
containing cosolvents and compare these to the absorbance of a baseline solution.
Toluene will be used as the transfer molecule with urea, NaCl, KSCN, and sucrose as
the respective cosolvents for each aqueous solution with a Toluene/water solution
providing the baseline. Students will then compare the effectiveness of the each
cosolvent based on data collected and explain reasons as to why the data fits the
theory provided.
Learning Objectives
1. Understand the hydrophobic effect and its relationship to protein folding.
a. It will be known that the students understand this concept if they
incorporate Student Question 5 into their discussion.
2. Understand the theory of transfer energy and be able to calculate its value
based upon absorbencies.
a. If the data and graph created for the results section is similar to the
expected results data graph (included), it will confirm that the students
understand how to calculate the change in transfer energy and
understand the theory.
3. Understand how cosolvents effect hydrophobic forces in a solution.
a. Question 4
4. Understand the meaning of positive and negative transfer energy values.
a. Questions 2 and 3
5. Understand the effect that each cosolvent has on hydrophobicity and why
this makes sense.
a. Questions 1, 4 and 5
Materials
Chemicals

Urea
Sodium Chloride
Sucrose
Potassium Thiocyanate
Toluene

Instruments/Supplies

Minimum of 96 sealable degradation resistant vials- 20 mL in volume

Parafilm
Quartz Cuvette
Distilled Water
Pipette/ Pipette Tips

Solution Beakers
Graduated Cylinders
Gloves and Googles
Hood
Laptop
Ocean Optics UV Spectrometer
LoggerPro
Kimwipes
Waste containers

Safety
Gloves should be worn at all times. All mixing and testing should be done under the
hood. KSCN and toluene are harmful by means of contact by skin and inhalation.
There should be labeled waste containers for both toluene and KSCN for the
students to use to dispose of chemicals with.
Preparation
Instruct students to install LoggerPro prior to lab and bring laptop when they come.
There are four cosolvents that need to be divided up between the classs lab
groups. For large classes, assign a single cosolvent to every group. For a smaller
classes, multiple cosolvents could be assigned to each lab group.
Stock solutions must be prepared by instructor prior to lab time. For each cosolvent
to be done in triplicate form, at least 120 mL of following molarities should be
prepared: Urea 2.5M, NaCl 5M, Sucrose 2M, and KSCN 1M. Students will perform
further dilution during the lab.
All supplies and protective equipment should be set out in a reasonable construct
prior to lab.
Procedure Notes
Reminders for the class before and during the lab:

Gloves and goggles are to be worn at all times.

All work will be performed under the hood.
KSCN and Toluene need to be disposed of in the proper waste containers.
Remind KSCN group that they will only put toluene in one set of the KSCN
samples.
Make sure containers are sealed with parafilm after adding Toluene.
Samples must be set for at least 24 hours but no longer than 3 days.
The three distilled water samples are needed as a baseline.
ONLY use a QUARTZ cuvette not a UV Cuvette.
Pull as much toluene off the samples as possible before taking samples to be
measured.
Make sure there are no Toluene bubbles in the line of sight for the lamp of
the UV Spectrometer as the absorbance will max out.

Make sure to rinse and wipe clean the Quartz cuvette between samples as
not to cross contaminate the cosolvents.
Make sure all students have all data at the end of lab to ensure they are able
to calculate the transfer energy fully.
Remind students that in the calculations, they need to use the water toluene
sample that correlates to the same trial of the specific cosolvent being
calculated.

Expected Results
The following tables and graphs are examples of what the instructor should expect
the students to obtain from the Lab.
Calculated change in transfer energy values:

Graph:

Answers to Student Questions

1. Look up the structure of toluene and explain why it was used as a
transfer molecule in this experiment instead of some other molecule.
(How does toluene relate to proteins?)
a. Because we are testing the properties of hydrophobicity and in specific
the hydrophobic effect on proteins, a hydrophobic molecule, such as
Toluene was needed to act as the hydrophobic amino acids on proteins.
2. When dealing with change in transfer energy, what does a negative
energy value mean about the cosolvent and how does it relate to the
change in transfer energy of water?
a. A negative value means that the hydrophobicity of the molecule was
decreased causing the toluene molecule to go into solution with a
smaller requirement of energy than water.
b. A positive value means that the hydrophobicity of the molecule was
increased causing the toluene molecule to resist entering solution with
a larger requirement of energy than water.
3. What do the positive and negative energy values mean for the
folding of proteins?
a. Proteins would unfold easier in a solution where the change in transfer
energy was negative. Proteins would fold more easily and be more
stable in a solution where the change in transfer energy was positive.

4. Explain at the molecular level, how hydrophobicity is effected by

each of the cosolvents. (Think bonding!)
a. The NaCl, KSCN, and sucrose each increase the surface tension of the
water by increasing the amount of hydrogen bonding occurring in the
solution. Toluene does not hydrogen bond well because it is a
hydrophobic molecule and is therefore forced to resist the solution.
Urea decreases the surface tension of the water by disrupting the
hydrogen bonding to the point that it no longer matters that the
toluene is hydrophobic and the molecule can go into solution with the
water and cosolvent.
5. Looking at the trend of each cosolvent from your graph, briefly
explain how the cosolvents would have an effect on the folding and
unfolding of proteins. (Which of the cosolvents studied would be
useful as a stabilizing agent for proteins? Which as a denaturant? )
a. NaCl and KSCN seem to stabilize proteins at about the same
effectiveness. Sucrose would also stabilize the folding of the proteins
but would not perform this task as well as NaCl or KSCN.
b. Urea would act as a denaturant and cause the protein to unfold.
Student Sheets
Excel document provided as a table for data.