Characterization of Protein Thermal Stability Using UV-Vis Spectroscopy

Significance of the Topic

Protein thermal stability is crucial in biopharmaceutical development, informing on structural integrity, formulation optimization, and storage conditions of therapeutic proteins.

Study Objectives and Overview

This study demonstrates a rapid and straightforward method for characterizing protein thermal stability and determining melting temperatures using the Agilent Cary 3500 UV-Vis spectrophotometer equipped with a Peltier temperature-controlled multicell module.

Methodology and Instrumentation

A native-state sample of Escherichia coli disulfide bond isomerase A (EcDsbA) at 0.6 mg/mL in phosphate buffer (50 mM NaCl, 1 mM EDTA, 100 mM sodium phosphate, pH 7.0) was analyzed. Absorbance scans from 200 to 400 nm (2 nm bandwidth, 0.1 s averaging) identified the 280 nm peak. Thermal melting experiments monitored absorbance at 280 nm using an ultra-microcell (70 μL, 10 mm pathlength). The Agilent Cary 3500 UV-Vis spectrophotometer with an eight-position, air-cooled Peltier multicell (0–110 °C) was controlled by Cary UV Workstation software. Thermal parameters included a ramp of 0.1 °C/min, data intervals of 0.1 °C, 2 s averaging, and smoothing/derivative filter size of 25 over 30–90 °C.

Main Results and Discussion

Simultaneous measurement of seven samples confirmed an absorbance maximum at 280 nm. Thermal melt curves revealed a structural transition above 65 °C. First-derivative analysis yielded an average Tm of 69.86 °C (n = 6) with a standard deviation of 0.16 °C. These values closely match literature reports obtained by circular dichroism.

Benefits and Practical Applications

The Cary 3500 system enables concurrent analysis of multiple replicates, enhancing throughput and consistency. Low-volume ultra-microcells minimize sample consumption without alignment requirements. Integrated temperature feedback ensures precise thermal control. This approach supports biopharmaceutical research, formulation screening, and quality control workflows.

Future Trends and Potential Applications

Advances may include high-throughput ligand-binding stability assays, integration with proteome-wide thermal profiling, and multi-wavelength kinetic studies. Enhanced automation and compliance features will further streamline analytical workflows in regulated environments.

Conclusion

By leveraging precise Peltier-based temperature control and simultaneous multi-sample measurement, the Agilent Cary 3500 UV-Vis spectrophotometer delivers accurate, reproducible melting temperatures rapidly and economically, aligning with established techniques while improving efficiency.

Reference

• Mohs RC, Greig NH. Drug discovery and development: Role of basic biological research. Alzheimers Dement (NY). 2017;11(3–4):651–657.
• Schenone M, et al. Target identification and mechanism of action in chemical biology and drug discovery. Nat Chem Biol. 2013;9(4):232–240.
• Gashaw I, et al. What makes a good drug target? Drug Discov Today. 2012;17(Suppl):S24–S30.
• Jahn TR, Radford SE. Folding versus aggregation: Polypeptide conformations on competing pathways. Arch Biochem Biophys. 2008;469(1):100–117.
• Mateus A, Määttä TA, Savitski MM. Thermal proteome profiling: Unbiased assessment of protein state through heat-induced stability changes. Proteome Sci. 2017;15:13.
• Ball KA, et al. An isothermal shift assay for proteome scale drug-target identification. Commun Biol. 2020;3:75.
• Greenfield NJ. Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions. Nat Protoc. 2006;1(6):2527–2535.
• Beychok S. Circular dichroism of biological macromolecules. Science. 1966;154(3754):1288–1299.
• Abd-Elghany M, Thomas MK. A review on differential scanning calorimetry technique and its importance in the field of energetic materials. Phys Sci Rev. 2018;3:20170103.
• Deangelis NJ, Papariello GJ. Differential scanning calorimetry. Advantages and limitations for absolute purity determinations. J Pharm Sci. 1968;57:1868–1873.
• Pantoliano MW, et al. High-density miniaturized thermal shift assays as a general strategy for drug discovery. J Biomol Screen. 2001;6(6):429–440.
• Ericsson UB, et al. Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal Biochem. 2006;357(2):289–298.
• Martin J, Bardwell JC, Kuriyan J. Crystal structure of the DsbA protein required for disulphide bond formation in vivo. Nature. 1993;365:464–468.
• Christensen S, et al. Structural and biochemical characterization of Chlamydia trachomatis DsbA reveals a cysteine-rich and weakly oxidising oxidoreductase. PLoS One. 2016;11(12):e0168485.
• Heras B, et al. Staphylococcus aureus DsbA does not have a destabilizing disulfide, a new paradigm for bacterial oxidative folding. J Biol Chem. 2008;283(7):4261–4271.