Microassay Thermal Denaturation of Ribonuclease A Probed by Circular Dichroism Spectroscopy

Download PDF August 17, 2022

Introduction

Circular dichroism (CD) is a useful technique to measure and analyze secondary structure and the thermal denaturation of proteins and nucleic acids in the far-UV region. Typically, a 1 mm pathlength rectangular cell is used for these measurements. However, this requires a sample volume of about 200 µL. In the case of samples where only very small volumes are available, JASCO now offers a new capillary cell for sample volumes smaller than 10 µL. The capillary cell also has a jacket for thermal ramping measurements, shown in Figure 1. CD measurements remain simple to perform and the capillary cells are inexpensive and disposable.

This application note demonstrates the use of a capillary cell and jacket for thermal ramping studies of ribonuclease A.

J-1500 Circular Dichroism Spectrometer
J-1500 Circular Dichroism Spectrometer

Experimental

Measurement conditions 
Data acquisition interval0.2°C
Path length0.5 mm (capillary cell), 1 mm (rectangular cell)
Spectral bandwidth1 nm
Rising temperature rate1°C/min
Measurement wavelength222 nm
Response time8 sec

1 mg/mL of ribonuclease A aqueous solution is drawn up into a capillary cell with a 0.5 mm optical pathlength, as seen in Figure 1. The cell is then inserted into the capillary jacket for thermal ramping CD measurements.

Capillary cell preparation diagram. For greater visibility of a sample in the photos above, a colored sample was used instead of ribonuclease A solution.
Figure 1. Capillary cell preparation diagram. For greater visibility of a sample in the photos above, a colored sample was used instead of ribonuclease A solution.

Keywords

Circular dichroism, capillary cell, microassay, thermal denaturation, ribonuclease A, Denatured Protein Analysis software, denaturation temperature, J-1500

Results

Figure 1 shows the thermal denaturation of ribonuclease A. The JASCO JWTDA-519 Denatured Protein Analysis software was used to calculate the denaturation temperature of ribonuclease A in both the capillary and rectangular cells. 59.4°C was calculated for the capillary cell which is in good agreement with 59.7°C obtained for the denaturation temperature in the rectangular cell.

Thermal denaturation curve of ribonuclease A measured at a wavelength of 222 nm. The green curve was obtained using the capillary cell while the blue curve was measured using the 1 mm rectangular cell.
Figure 2. Thermal denaturation curve of ribonuclease A measured at a wavelength of 222 nm. The green curve was obtained using the capillary cell while the blue curve was measured using the 1 mm rectangular cell.

Conclusion

This application note demonstrates that the denaturation temperature values, along with the thermal denaturation curves shown in Figure 2, illustrate that the microassay of ribonuclease A using the capillary cell and jacket can be carried out with high accuracy.

Note: The MSD-462 microsampling disc can be used for spectral scanning measurements on sample volumes between 2 and 10 µL. The MSD-462 applications are shown in the following Application Notes: 260-CD-0011 and 260-CD-0019.

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About the Author

Leah Pandiscia received her PhD from Drexel University where she studied Biophysical Chemistry. She is a Spectroscopy Applications Scientist.