Application case of differential scanning calorimeter for measuring protein thermal stability

In drug development,Differential scanning calorimeterCommonly used in drug screening and optimization processes, especially in the development of compounds that require binding to specific proteins. This method can measure the thermal behavior of proteins when subjected to heat, especially the amount of heat released or absorbed when their structure changes. DSC can provide important information about protein folding and stability, which is very valuable for fields such as drug design, biotechnology, and disease research. By comparing the DSC curves of proteins with and without bound drugs, it is possible to evaluate how drug phase selectivity affects the thermal stability of proteins.

In the field of biopharmaceuticals, DSC is often used as a quality control method to ensure the stability and integrity of collagen drugs throughout the entire production and storage process.

The DSC method can not only provide information on protein thermal stability, but also assist in studying protein folding dynamics and secondary structure. This is achieved by analyzing different transitions on the DSC curve, which may correspond to different protein structures or other reactions.

　　1、 Experimental instruments and settings

DZ-DSC300C Differential Scanning Calorimeter

2. High purity nitrogen gas

3. Collagen protein of the tested sample

4. Standard aluminum crucible dish

5. Standard tweezers

　2、 Experimental principle and steps

　　1. Principle

DSC is a technique for measuring the absorption or release of heat by a sample during heating or cooling. By comparing the difference in heat flux between the sample and the reference material under temperature changes, the thermal properties of the sample can be obtained. Collagen denaturation caused by heating is called thermal denaturation. Denaturation can cause changes in physical, chemical, and biological properties. The various biological functions exhibited by proteins in life activities depend entirely on their specific conformation. Once this specific conformation is disrupted, their biological functions will disappear. Therefore, studying the thermal denaturation of collagen under different conditions is of great significance. Differential scanning calorimetry can directly provide the temperature and energy changes during protein thermal denaturation, and is a very effective method for studying the conformational changes and structural stability of collagen.

　　2. Operation steps　

2.1 Take 10-15mg of the sample and weigh it in a crucible.

2.2 Install and adjust the gas flow rate according to the instrument's operating manual.

2.3 Write a temperature rise program and conduct temperature rise tests within the range of 70-90 ° C at a rate of 5 ℃/min.

2.4 Record experimental data and analyze the results.

Through experimental analysis of the graph, it can be directly observed that the melting point (TM) of the protein is 46 ℃ and a temperature point at which denaturation begins, indicating that stability is disrupted. For proteins, a enthalpy value of 50J/g means the total amount of heat required to induce protein folding, representing an endothermic process. Due to the exposure of proteins to elevated temperatures in DSC experiments, they begin to undergo thermal denaturation, accompanied by the breaking of non covalent bonds.

This experiment can also determine whether the protein is inactivated. In DSC technology, Δ Hcat is determined only by the area of the integrated DSC thermal transition peak curve, while Δ Hv is determined only by the shape of the thermal transition peak curve. The sharper the peak shape, the larger the Δ HvH, and vice versa. Using Δ Hca/Δ Hvn can help us estimate whether a significant portion of proteins are inactive. If Δ Hcal is significantly lower than Δ HvH, it indicates that a significant portion of the protein has been inactivated.

In summary, analyzing the data in DSC can help us understand the denaturation temperature point of proteins and determine whether proteins have become inactive.