1. 1
Thermal Denaturation of a
Protein
Andrew LeSage
CH3541-L01
March 03, 2015

2. 2
Abstract
The first goal of this experiment was to determine the spectral properties of phenylalanine,
tryptophan, and tyrosine. The peak absorbance of phenylalanine was found to be 257 nm and the
peak absorbance for tryptophan and tyrosine were 279 nm and 277 nm, respectively. Next, the
thermodynamic properties of Lysozyme in acetate buffer (pH 4.0) and glycine HCl buffer (pH
2.5) at both low and high temperatures were analyzed. In acetate buffer, the equilibrium constant
for the denaturation of Lysozyme was found to be 0.652, the Gibb’s free energy was found to
be 1164.47
𝐽
𝑚𝑜𝑙
, and the enthalpy of denaturation was found to be −194.92 𝑘𝐽. In glycine HCl
buffer, the equilibrium constant for the denaturation of Lysozyme was found to be -2.23, making
calculations for Gibb’s free energy and enthalpy impossible. By comparing Tm values for each
trial, it was observed that Lysozyme is more stable at higher temperatures, when the pH is 4.0
instead of pH 2.5.
Background
This experiment is going to observe the denaturation of Lysozyme using the amino acid residues
of phenylalanine (F), tryptophan (W), and tyrosine (Y). The spectral properties of these proteins’
native and denatured states are going to be used to observe Lysozyme as a function of
temperature and pH. The denaturation of these proteins into native and denatured states can be
shown in a model as shown in equation 1.
[1]
𝑁 ↔ 𝐷
UV-Vis spectroscopy was used to monitor progress of the denaturation of Lysozyme. Different
molecules absorb light at different wavelengths. UV-Vis spectroscopy generates an absorbance
spectra that shows absorbance changes as a function of wavelength. Observed changes in
absorption can then be used to monitor the progress of structural changes that are occurring
during denaturation. [1] Using UV-Vis spectroscopy to monitor the denaturation of Lysozyme is
possible because the aromatic side chains of phenylalanine, tryptophan, and tyrosine can absorb
UV range light. [2]
The following two equations, equations 2 and 3, can find the linear baselines for the native and
denatured states based off of a graph of the denaturation of a protein using the model in equation
1.
[2]
𝑦 𝑁,𝑇 = 𝑚 𝑁 𝑇 + 𝑏 𝑁
[3]
𝑦 𝐷,𝑇 = 𝑚 𝐷 𝑇 + 𝑏 𝐷
Where mn and bn are the slope and y-intercept of the native baseline, mD and bD represent the
denatured base line, and T is the absolute temperatures. Both of these equations are functions of
temperatures.

3. 3
The fraction of the denatured protein, αT, can be compared at different signals, AT, at various
temperatures of the native and denatured baselines.
[4]
𝛼 𝑇 =
(𝐴 𝑇 − 𝑦 𝑁,𝑇)
(𝑦 𝐷,𝑇 − 𝑦 𝑁,𝑇)
KD,T which is the equilibrium constant for the denaturation of the protein can be found by
equation 5.
[5]
𝐾 𝐷,𝑇 =
[𝐷]
[𝑁]
=
𝛼 𝐷,𝑇
1 − 𝛼 𝐷,𝑇
[D] is the concentration of the protein in the denatured state and [N] is the amount of protein in
the native state.
The Gibb’s free energy of the denaturation of the protein can be is shown in equation 6.
[6]
∆𝐺 𝐷,𝑇 = −𝑅𝑇𝑙𝑛𝐾 𝐷,𝑇
ΔG is dependent on the stability of the native and denatured state of the proteins.
The van’t Hoff equation can be used to find ΔHD which is the enthalpy of the denaturation.
Equation 7 shows this.
[7]
∆𝐻 𝐷 = −4𝑅𝑇𝐷
2
(
𝛿𝛼
𝛿𝑇
) 𝑇,𝐷
(
𝛿𝛼
𝛿𝑇
) is the slope of the line of the graph and is used with TD.
Methods
Amino Acid Preparation
Approximately 1.0-mL samples of 7.5 mM phenylalanine, 0.18 mM tryptophan, and 0.13 mM
tyrosine were prepared using the acetate buffer (pH 4.0) provided by the TA. The samples were
stored on ice until they were analyzed.
Peptide Spectra
The Peltier Control was set to 25o
C and the UV-VIS spectrum of each of the amino acids was
obtained using the Perkin Elmer Lambda 35 UV-VIS spectrometer. The absorbance was
recorded from 220 to 400nm.

4. 4
Difference Spectra
Next, a sample of 0.3 mg/mL Lysozyme in glycine buffer (pH 2.5) was obtained from the TA.
The sample was allowed to equilibrate in the spectrometer for 2 minutes, at 25o
C. Absorbance
was recorded from 220 to 400nm, generating the native structure spectra. After spectra was
recorded, the temperature was raised to approximately 90o
C and the sample was allowed to
equilibrate for approximately 5 minutes. The spectra was recorded again under the same
wavelength conditions, yielding the denatured structure spectra. A second group repeated this
experiment for the same protein in acetate buffer (pH 4.).
Thermal Denaturation Curves
Absorbance data of 0.3 mg/ml Lysozyme in pH 2.5 glycine HCl buffer was collected at 301 nm
from 25o
C to 85o
C. Between each measurement, temperature was raised 2o
C and the sample was
allowed to equilibrate for 2 minutes before absorbance was measured. At maximum absorbance
changes, temperature was raised 1o
C between measurements. The sample was still allowed to
equilibrate for 2 minutes between measurements. A second group repeated this procedure for 0.3
mg/mL Lysozyme in pH 4.0 acetate buffer.
Results
Peptide Spectra
Figure 1. UV absorbance at different wavelengths is plotted for phenylalanine, tryptophan, and
tyrosine in acetate buffer, pH 4. Peak wavelength is marked
257 nm 279 nm
277nm
-0.1
0.1
0.3
0.5
0.7
0.9
1.1
1.3
1.5
240 250 260 270 280 290 300 310 320
O.D.
Wavelength (nm)
Peptide Spectra, Acetate Buffer (pH 4.0)
Phenylalanine (F), 7.5 mM
Tryptophan (W), 0.18 mM
Tyrosine (Y), 0.13 mM

5. 5
Difference Spectra
Figure 2. UV absorbance of 0.3 mg/mL native and denatured Lysozyme in glycine buffer (pH
2.5) is plotted on the left. On the right, a graph of the difference in UV absorbance between
native and denatured Lysozyme is plotted.
Figure 3. UV absorbance of 0.3 mg/mL native and denatured Lysozyme, in acetate buffer (pH
4.0) is plotted on the left. On the right, a graph of the difference in UV absorbance between
native and denatured Lysozyme is plotted.
280 nm
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
240 260 280 300 320 340
O.D.
Wavelength, nm
Protein Spectra
Denatured
Native
UV Absorbance of 0.3 mg/mL Lysozyme at pH 2.5
281 nm
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
240 260 280 300 320 340
O.D.
Wavelength, nm
Protein Spectra
Denatured
Native
UV Absorbance of 0.3 mg/mL Lysozyme at pH 4.0
254 nm
0
0.02
0.04
0.06
0.08
0.1
240 260 280 300 320 340
O.D.
Wavelength, nm
pH 2.5 difference
253 nm
-0.04
-0.02
0
0.02
0.04
0.06
0.08
240 290 340
O.D.
Wavelength, nm
pH 4.0 Difference

6. 6
Thermal Denaturation Curves
Figure 4. Thermal denaturation curve of Lysozyme at pH 2.5 is shown above.
Figure 5. Thermal denaturation curve of 0.3 mg/mL lysozyme at pH 4.0 is shown above.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
24 44 64 84
FractionofDenatured
Temperature (°C)
Thermodenaturation Curve of 0.3 mg/ml Lysozyme at 310
nm, pH 2.5 Glycine HCl Buffer
normalized
Linear (Native
Fit)
Linear
(Transition)
Linear
(denatured)
0
0.2
0.4
0.6
0.8
1
1.2
20 40 60 80 100
FractionofDenaturaiton
Temperature (°C)
Thermodenaturation Curve of 0.3 mg/mL Lysozyme
at 301 nm, pH 4.0 Acetate buffer
normalized
Linear
(Native)
Linear
(Transition)
Linear
(Denatured)

7. 7
Calculations
Lysozyme at pH 4.0
𝑦 𝑁,𝑇 = 𝑚 𝑁 𝑇 + 𝑏 𝑁 = 0.0132(25°𝐶 ) − 0.3925 = −𝟎. 𝟎𝟔𝟐𝟓
𝑦 𝐷,𝑇 = 𝑚 𝐷 𝑇 + 𝑏 𝐷 = 0.013(25°𝐶) − 0.0367 = 𝟎. 𝟐𝟖𝟖𝟑
𝛼 𝑇 = (
𝐴 𝑇 − 𝑦 𝑁,𝑇
𝑦 𝐷,𝑇 − 𝑦 𝑁,𝑇
) = (
0.076 − (−0.0625)
0.2883 − (−0.0625)
) = 𝟎. 𝟑𝟗𝟒𝟖
𝐾 𝐷,𝑇 =
[𝐷]
[𝑁]
=
𝛼 𝐷,𝑇
1 − 𝛼 𝐷,𝑇
=
0.3948
1 − 0.3948
= 𝟎. 𝟔𝟓𝟐
∆G 𝐷,𝑇 = −𝑅𝑇𝑙𝑛𝐾 𝐷,𝑇 = − (8.314
𝐽
𝑚𝑜𝑙 ∗ 𝐾
) (298𝐾)𝑙𝑛(0.625) = 𝟏𝟏𝟔𝟒. 𝟒𝟕
𝑱
𝒎𝒐𝒍
∆𝐻 𝐷 = −4𝑅𝑇𝐷
2
(
𝛿𝛼
𝛿𝑇
)
𝑇𝐷
= −4 (8.314
𝐽
𝑚𝑜𝑙 ∗ 𝐾
) (298𝐾)2(0.066) = −𝟏𝟗𝟒. 𝟗𝟐 𝒌𝑱
Lysozyme at pH 2.5
𝑦 𝑁,𝑇 = 𝑚 𝑁 𝑇 + 𝑏 𝑁 = 0.0088(25°𝐶) + 0.0245 = 𝟎. 𝟐𝟒𝟒𝟓
𝑦 𝐷,𝑇 = 𝑚 𝐷 𝑇 + 𝑏 𝐷 = 0.0147(25°𝐶) − 0.3333 = 𝟎. 𝟑𝟒𝟐
𝛼 𝑇 = (
𝐴 𝑇 − 𝑦 𝑁,𝑇
𝑦 𝐷,𝑇 − 𝑦 𝑁,𝑇
) = (
0.068 − 0.2445
0.342 − 0.2445
) = 𝟏. 𝟖𝟏
𝐾 𝐷,𝑇 =
[𝐷]
[𝑁]
=
𝛼 𝐷,𝑇
1 − 𝛼 𝐷,𝑇
=
1.81
1 − 1.81
= −𝟐. 𝟐𝟑
Gibb’s Free Energy and Enthalpy cannot be calculated because the calculated K value has a
negative sign.
Discussion
In this experiment, the Tm of the Lysozyme varied when pH was adjusted between 2.5 and 4.0.
At pH of 2.5, the Tm was at 54°C whereas it was 69°C, at pH 4.0. This demonstrates that the
structure of lysosome is more stable at a higher pH. Structurally, this makes sense because the
amino acid side chains that are present on lysosome are aspartic and glutamic acid. Both acids
are in their negatively charged states at pH 4.0 or higher. When the pH is at 2.5, the amino acids
are protonated—not having a negative charge that is capable of stabilizing the protein. The
absence of negatively charged stabilizing amino acid side chains on lysosome at lower pH
explains why amino acids have a lower Tm under such conditions. Lysosome in higher pH
conditions remains in the native conformation longer than lysosome at lower pH.

8. 8
Conclusion
This experiment can be considered successful. The thermodynamic properties of Lysozyme in
acetate buffer (pH 4.0) and glycine HCl buffer (pH 2.5) at both low and high temperatures were
analyzed. In acetate buffer, the equilibrium constant for the denaturation of Lysozyme was found
to be 0.652, the Gibb’s free energy was found to be 1164.47
𝐽
𝑚𝑜𝑙
, and the enthalpy of
denaturation was found to be−194.92 𝑘𝐽. In glycine HCl buffer, the equilibrium constant for the
denaturation of Lysozyme was found to be -2.23, making calculations for Gibb’s free energy and
enthalpy impossible. By comparing Tm values for each trial, it was observed that Lysozyme is
more stable at higher temperatures, when the pH is 4.0 instead of pH 2.5. If this experiment were
conducted again, the thermodynamic properties of a variety of other enzymes would be tested to
better understand how these properties vary when temperature is adjusted.
References
1. Misra, P., & Dubinskii, M. A. (Eds.). (2002). Ultraviolet spectroscopy and UV lasers.
CRC Press.
2. “Protein Study I: Thermal Analysis of a Protein”, CH3541 Spring 2015 web pages,
https://mtu.instructure.com/courses/979790/assignments/4256377, “PRE: Protein
Analysis Part I: Thermal Analysis”. Found Mar. 18, 2015.