1

Thermal Denaturation of a

Protein

Andrew LeSage

CH3541-L01

March 03, 2015

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]

𝑦𝑁,𝑇 = 𝑚𝑁𝑇 + 𝑏𝑁

[3]

𝑦𝐷,𝑇 = 𝑚𝐷𝑇 + 𝑏𝐷

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.

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The fraction of the denatured protein, αT, can be compared at different signals, AT, at various

temperatures of the native and denatured baselines.

[4]

𝛼𝑇 =(𝐴𝑇 − 𝑦𝑁,𝑇)

(𝑦𝐷,𝑇 − 𝑦𝑁,𝑇)

KD,T which is the equilibrium constant for the denaturation of the protein can be found by

equation 5.

[5]

𝐾𝐷,𝑇 = [𝐷]

[𝑁]=

𝛼𝐷,𝑇

1 − 𝛼𝐷,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]

∆𝐺𝐷,𝑇 = −𝑅𝑇𝑙𝑛𝐾𝐷,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]

∆𝐻𝐷 = −4𝑅𝑇𝐷2(

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 25oC 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.

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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 25oC. Absorbance

was recorded from 220 to 400nm, generating the native structure spectra. After spectra was

recorded, the temperature was raised to approximately 90oC 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 25oC to 85oC. Between each measurement, temperature was raised 2oC and the sample was

allowed to equilibrate for 2 minutes before absorbance was measured. At maximum absorbance

changes, temperature was raised 1oC 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

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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

00.10.20.30.40.50.60.70.80.9

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

00.10.20.30.40.50.60.70.80.9

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

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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

Fra

ctio

nof

Den

ature

d

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

Fra

ctio

n o

f D

enat

ura

iton

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)

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Calculations

Lysozyme at pH 4.0

𝑦𝑁,𝑇 = 𝑚𝑁𝑇 + 𝑏𝑁 = 0.0132(25°𝐶 ) − 0.3925 = −𝟎. 𝟎𝟔𝟐𝟓

𝑦𝐷,𝑇 = 𝑚𝐷𝑇 + 𝑏𝐷 = 0.013(25°𝐶) − 0.0367 = 𝟎. 𝟐𝟖𝟖𝟑

𝛼𝑇 = (𝐴𝑇 − 𝑦𝑁,𝑇

𝑦𝐷,𝑇 − 𝑦𝑁,𝑇) = (

0.076 − (−0.0625)

0.2883 − (−0.0625)) = 𝟎. 𝟑𝟗𝟒𝟖

𝐾𝐷,𝑇 =[𝐷]

[𝑁]=

𝛼𝐷,𝑇

1 − 𝛼𝐷,𝑇=

0.3948

1 − 0.3948= 𝟎. 𝟔𝟓𝟐

∆G𝐷,𝑇 = −𝑅𝑇𝑙𝑛𝐾𝐷,𝑇 = − (8.314𝐽

𝑚𝑜𝑙 ∗ 𝐾) (298𝐾)𝑙𝑛(0.625) = 𝟏𝟏𝟔𝟒. 𝟒𝟕

𝑱

𝒎𝒐𝒍

∆𝐻𝐷 = −4𝑅𝑇𝐷2 (

𝛿𝛼

𝛿𝑇)

𝑇𝐷= −4 (8.314

𝐽

𝑚𝑜𝑙 ∗ 𝐾) (298𝐾)2(0.066) = −𝟏𝟗𝟒. 𝟗𝟐 𝒌𝑱

Lysozyme at pH 2.5

𝑦𝑁,𝑇 = 𝑚𝑁𝑇 + 𝑏𝑁 = 0.0088(25°𝐶) + 0.0245 = 𝟎. 𝟐𝟒𝟒𝟓

𝑦𝐷,𝑇 = 𝑚𝐷𝑇 + 𝑏𝐷 = 0.0147(25°𝐶) − 0.3333 = 𝟎. 𝟑𝟒𝟐

𝛼𝑇 = (𝐴𝑇 − 𝑦𝑁,𝑇

𝑦𝐷,𝑇 − 𝑦𝑁,𝑇) = (

0.068 − 0.2445

0.342 − 0.2445) = 𝟏. 𝟖𝟏

𝐾𝐷,𝑇 =[𝐷]

[𝑁]=

𝛼𝐷,𝑇

1 − 𝛼𝐷,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.

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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.