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Home / Applications / Fluorescence Measurement of Heat-Denatured Lysozyme

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  • Technique

Fluorescence Measurement of Heat-Denatured Lysozyme

By Heather Haffner

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August 25, 2022

Introduction

Experiments studying the denaturation of proteins are generally made using circular dichroism. However, proteins contain aromatic amino acids (AAA) will fluoresce when excited with UV light. During heat denaturation of proteins, the secondary structure of the protein will change and the aromatic amino-acid residues will change slightly which can be detected by fluorescence.

FP-6500 spectrofluorometer

Experimental

Emission (EM) spectra of lysozyme using an excitation (EX) of 280 nm are measured while controlling the temperature, to examine the relation between temperature and the fluorescence spectrum. The FP-6500 spectrofluorometer with a ETC-272 Peltier thermostatted cell holder are used for the measurements in this experiment, using the instrument parameters outlined below.

Parameters

A buffered aqueous solution of 0.1mg/mL of lysozyme was used as the sample, the measurement performed by stirring the sample with a magnetic stirrer to ensure even sample temperatures in the cell. A temperature ramping rate of 1°C/min was controlled by the Peltier cell holder. Spectra were measured 60 seconds after reaching the set temperature.

Results

The graph below (Figure 1) shows the change in intensity vs. temperature at 340 nm. Lysozyme is known to denature at a temperature of 70°C; however the graph of fluorescence vs. temperature shows only a decrease in intensity as the temperature increases. By contrast, the EM Spectra from 40 to 90 degrees plotted using a contour view demonstrates a transition of the spectra at 70 degrees (Figure 2).

Figure 1. Temperature dependence of Lysozyme in aqueous solution at EX 280 nm, EM 340 nm
Figure 2. Contour view of EM spectra from 40 to 90 degrees.

The EM spectra at room temperature have a maxima at 340 nm with a corresponding maxima at 348 nm for 90 degrees. A plot of the intensity ratios for the two wavelengths versus temperature results in a graph that demonstrates a heat-denaturation at 70°C (Figure 3), in agreement with literature values.

Figure 3. Intensity ratio of 340/348 nm vs. temperature.

Required Products and Software

  • FP-8200/8300/8500/8600/8700 Spectrofluorometer
  • ETC-814/815 Water-cooled Peltier Thermostatted Cell Holder with Stirrer, or PTC-818 Water-cooled Peltier Thermostatted 4-position Automatic Cell Changer with Stirrer
  • FWTS-872 Temperature Interval Scan Measurement program
This document has been prepared based on information available at the time of publication and is subject to revision without notice. Although the contents are checked with the utmost care, we do not guarantee their accuracy or completeness. JASCO Corporation assumes no responsibility or liability for any loss or damage incurred as a result of the use of any information contained in this document. Copyright and other intellectual property rights in this document remain the property of JASCO Corporation. Please do not attempt to copy, modify, redistribute, or sell etc. in whole or in part without prior written permission.

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About the Author

JASCO Application Note

Fluorescence Measurement of Heat-Denatured Lysozyme

Introduction

Experiments studying the denaturation of proteins are generally made using circular dichroism. However, proteins contain aromatic amino acids (AAA) will fluoresce when excited with UV light. During heat denaturation of proteins, the secondary structure of the protein will change and the aromatic amino-acid residues will change slightly which can be detected by fluorescence.

FP-6500 spectrofluorometer

Experimental

Emission (EM) spectra of lysozyme using an excitation (EX) of 280 nm are measured while controlling the temperature, to examine the relation between temperature and the fluorescence spectrum. The FP-6500 spectrofluorometer with a ETC-272 Peltier thermostatted cell holder are used for the measurements in this experiment, using the instrument parameters outlined below.

Parameters

A buffered aqueous solution of 0.1mg/mL of lysozyme was used as the sample, the measurement performed by stirring the sample with a magnetic stirrer to ensure even sample temperatures in the cell. A temperature ramping rate of 1°C/min was controlled by the Peltier cell holder. Spectra were measured 60 seconds after reaching the set temperature.

Results

The graph below (Figure 1) shows the change in intensity vs. temperature at 340 nm. Lysozyme is known to denature at a temperature of 70°C; however the graph of fluorescence vs. temperature shows only a decrease in intensity as the temperature increases. By contrast, the EM Spectra from 40 to 90 degrees plotted using a contour view demonstrates a transition of the spectra at 70 degrees (Figure 2).

Figure 1. Temperature dependence of Lysozyme in aqueous solution at EX 280 nm, EM 340 nm
Figure 2. Contour view of EM spectra from 40 to 90 degrees.

The EM spectra at room temperature have a maxima at 340 nm with a corresponding maxima at 348 nm for 90 degrees. A plot of the intensity ratios for the two wavelengths versus temperature results in a graph that demonstrates a heat-denaturation at 70°C (Figure 3), in agreement with literature values.

Figure 3. Intensity ratio of 340/348 nm vs. temperature.

Required Products and Software

  • FP-8200/8300/8500/8600/8700 Spectrofluorometer
  • ETC-814/815 Water-cooled Peltier Thermostatted Cell Holder with Stirrer, or PTC-818 Water-cooled Peltier Thermostatted 4-position Automatic Cell Changer with Stirrer
  • FWTS-872 Temperature Interval Scan Measurement program
This document has been prepared based on information available at the time of publication and is subject to revision without notice. Although the contents are checked with the utmost care, we do not guarantee their accuracy or completeness. JASCO Corporation assumes no responsibility or liability for any loss or damage incurred as a result of the use of any information contained in this document. Copyright and other intellectual property rights in this document remain the property of JASCO Corporation. Please do not attempt to copy, modify, redistribute, or sell etc. in whole or in part without prior written permission.
28600 Mary’s Court, Easton, MD 21601 USA • (800) 333-5272 • Fax: (410) 822-7526 • jascoinc.com/applications

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