Protein Thermal Stability Analysis

Protein thermal stability refers to the ability of a protein polypeptide chain to change shape under the influence of temperature, mainly reflected in the unique chemical properties and spatial conformation changes of the polypeptide chain when the temperature changes. The smaller the deformation, the higher the thermal stability. In the fields of biotechnology, drug research and development, and the food industry, protein thermal stability analysis is of great significance. The thermal stability of proteins is generally represented by the denaturation midpoint temperature (Tm), which is the temperature at which 50% of the protein is unfolded. The commonly used methods to measure the Tm value of proteins include the following:

 

1. Circular Dichroism (CD)

CD is a widely used method in protein structure research. The principle is that there is a difference in the absorption of left-handed and right-handed circularly polarized light by proteins. The spectral characteristics of the far ultraviolet region (190~260nm) can be used to quickly analyze the secondary structure of the protein in the solution, and then analyze and identify the type of the protein's tertiary structure. In addition, the Tm of the protein can be obtained by measuring the average residual molar ellipticity [θ] of the protein at different temperatures.

 

Features: Suitable for measuring the thermal stability of dilute solutions, high concentration solutions may make the results inaccurate due to the interaction between protein molecules; the operation is relatively simple, and the cost is lower.

 

2. Differential Scanning Calorimetry (DSC)

The heat difference produced by simultaneously heating or cooling the reference object and the sample to maintain the same temperature is measured, thereby calculating the Tm value of the protein.

 

Features: DSC can provide direct heat change data, which is accurate in quantification and easy to operate; however, it has a low detection throughput and takes a long time.

 

3. Differential Scanning Fluorescence (DSF)

Also known as thermal fluorescence. According to the different sources of fluorescence, it is divided into DSF based on endogenous dye fluorescence and DSF based on exogenous fluorescence.

 

DSF based on endogenous fluorescence: Its principle is mainly based on the fluorescence characteristics of specific amino acids (such as tryptophan and tyrosine) in proteins. The fluorescence intensity of these amino acids is closely related to the microenvironment in which they are located. Therefore, when the structure of the protein changes, the fluorescence signal of these amino acids will also change.

 

Features: No need for complicated sample treatment or labeling steps, the experiment is relatively simple; but not all proteins contain enough fluorescent groups, and they may be affected by other groups.

 

4. DSF Based on Exogenous Dye Fluorescence

The principle is to use a dye that can interact with the hydrophobic groups inside the protein as the fluorescence source. When the protein is heated and denatured, the hydrophobic groups are exposed, and the hydrophobic groups bind to the affinity dye to produce a fluorescent signal. The instrument monitors the change in fluorescence intensity to determine the Tm value.

 

Features: Suitable for high-throughput screening, the signal strength is controllable, and the sensitivity and accuracy are relatively high; however, the added exogenous dye may affect the structure and function of the protein, and the operation is more complicated.

 

In addition to the above methods, there are some other analysis methods, such as ultracentrifugation dispersion deposition method, PCR instrument heating method, etc. These methods have specific applicable ranges and limitations, so in actual applications, it is necessary to consider multiple factors. , Choose the most suitable method to analyze the thermal stability of proteins.

 

MtoZ Biolabs is equipped with the MicroCal VP-Capillary DSC system from Malvern Panalytical, which can efficiently and accurately analyze the thermal stability of biological products. In addition, this automated, integrated high-throughput platform can also be applied to the identification and screening of biological drugs, ligand interactions, and other research, and we welcome your consultation.