Thermal Melting Analysis of Multiple Samples using High-Throughput Circular Dichroism (HTCD)

Download PDF May 26, 2017

Introduction

Thermal denaturation CD measurements provide important information regarding a protein’s thermodynamic properties as well as secondary structure characteristics. The J-1500 HTCD system enables researchers to automate not only CD spectral measurements and obtain secondary structure estimations, but also variable temperature measurements and thermal denaturation analysis of multiple samples.

This application note demonstrates the use of the J-1500 CD spectrometer and High-Throughput system to obtain variable temperature measurement data and secondary structure estimations using the Thermal Denaturation Analysis program.

J-1500 CD Spectrometer

Experimental

Measurement conditions   
Data interval0.2 °CWavelength222 nm
Spectral bandwidth0.2 nmPath length2 nm

0.2 mg/mL of human serum albumin (HSA), lysozyme and riboneclease A were prepared in H20.

Keywords

J-1500, circular dichroism, proteins, melting temperature analysis, HTCD, ASU-800 Auto Sampler, JFLC-498 Peltier flow cell, pharmaceuticals, biochemistry

Results

The flow cell for the HTCD system is designed to prevent the influence of temperature increases on baseline measurements. Figure 1 shows that the CD signal of water used as a blank solvent is stable from 20°C to 90°C.

Figure 1. CD stability of the HTCD system shown by using a water sample as a blank

The analysis of the CD results from the variable temperature measurements of HSA, lysozyme, and ribonuclease A are shown below (Figure 2). The thermodynamic properties were calculated using the Thermal Denaturation Program and are shown in Table 1.

Figure 2. Thermal Denaturation Analysis program displaying CD measurements from three protein samples

Table 1. Thermodynamic parameters of three proteins measured using the HTCD system and calculated using the Thermal Denaturation Analysis program

Sample name
Tm (°C)
ΔH (kcal/mol)
ΔS (kcal/mol*K)
Human serum albumin73.546.40.134
Lysozyme74.565.80.189
Ribonuclease A58.4135.40.408

Conclusion

This application note illustrates that the J-1500 CD spectrometer, coupled with the HTCD system can provide highly precise measurements for multiple protein samples. Additionally, thermodynamic parameters can be easily calculated using the Thermal Denaturation Analysis program.

About the Author

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