Total fluorescence and fluorescence scanning
Intrinsic fluorescence can be measured on the J-1500 or J-1700 CD spectrometers, with either the Peltier thermostatted single cell holders (PTC-510 and PTC-517) or automatic 6-position Peltier turret cell changer (MPTC-513). A simple low-cost system for detection of total fluorescence is available using a secondary detector and high-pass filters (FDT-538 and FST-470), allowing the user to select the excitation wavelength while detecting the emission at the wavelengths above the cut-off filter. This allows for simple, yet sensitive, detection of fluorescence changes during titration or thermal ramp experiments.
![](https://www.jasco-global.com/wp-content/uploads/2023/10/FDT-538_540x450px.png)
![](https://www.jasco-global.com/wp-content/uploads/2023/10/FMO-522_540x500px.png)
Alternatively, fluorescence data can be acquired by using the optional scanning emission monochromator (FMO-522) and emission detector (FDT-538). Excitation and fluorescence emission spectra can be scanned by fixing the emission or excitation wavelengths, respectively.
• Fluorescence scanning can be coupled with the titration and thermal ramping capabilities
• With the MPTC-513, CD and fluorescence data can be collected, simultaneously or separately, on up to six samples
• With the HTCD Plus, high-throughput fluorescence scanning is also available.
Application
Thermal denaturation of lysozyme measured with CD and fluorescence spectroscopies
Lysozyme, a globular protein found in the white of a hen’s egg, is a model protein used to investigate the denaturation of proteins at high temperature. The secondary structure of lysozyme comprises about 38 % α-helix and 10 % β-sheet.
Chicken egg-white lysozyme (1 mg) was dissolved in 15 mL of deionized water. The thermal denaturation of the protein was monitored using the J-1500 CD spectrophotometer equipped with an MPTC-513 automatic 6-position Peltier turret cell changer and an FMO-522 emission monochromator for detection of fluorescence. CD and fluorescence spectra were automatically measured at 5 °C intervals from 20 to 95 °C. After the final measurement at 95 °C, the sample temperature was returned to 20 °C and a final spectrum was collected.
![](https://www.jasco-global.com/wp-content/uploads/2023/10/ThermalDenaturation_1_540x350px.png)
![](https://www.jasco-global.com/wp-content/uploads/2023/10/ThermalDenaturation_2_540x350px.png)
Thermal denaturation measured with CD spectroscopy
Left: As the temperature increases, the intensity of the CD spectra decreases and the minimum at 208 nm blue-shifts to 203 nm.
Right: Upon completion of the melt, the temperature is re-equalibrated at the initial 20 °C. Comparison of the CD spectra measured at 20 °C before and after the melt demonstrates that while the protein does refold, it does not recover its original structure. (Green: 20 °C initial / Blue: 95 °C / Red: 20 °C final)
![](https://www.jasco-global.com/wp-content/uploads/2023/10/ThermalDenaturation_3_540x350px.png)
![](https://www.jasco-global.com/wp-content/uploads/2023/10/ThermalDenaturation_4_540x350px.png)
Fluorescence data for the thermal denaturation of Iysozyme from 20 to 95 °C
Left: As the protein undergoes thermal denaturation, the fluorescence decreases in intensity and the emission maximum red-shifts from 338 to 347 nm. As with the CD data, the largest shift occurs between 75 and 80 °C.
Right: A comparison of the protein fluorescence spectra measured at 20 °C before and after thermal denaturation supports the CD results, which indicate that the Iysozyme structure does not return to its initial native state after denaturation. (Green: 20 °C initial / Blue: 95 °C / Red: 20 °C final)