What factors affect protein stability?

Proteins are relatively fragile molecules that are susceptible to denaturation (loss of structure due to unfolding) during characterization and purification. Main factors that affect protein stability include: 

1. High temperature – Higher temperatures increase kinetic energy, which causes molecules to vibrate rapidly and forcefully. This results in a disruption of the hydrogen bonds and other nonpolar hydrophobic interactions, leading to the denaturation of the protein. Maintaining optimum temperature can keep proteins stable and minimize temperature induced denaturation. 

2. Protease Activity – Proteolytic enzymes known as proteases are released into the buffer environment during cellular disruption. These proteases cleave the protein, resulting in loss of stability and protein denaturation. Protease activity can be reduced and protein stability can be maintained by using rapid purification procedures, keeping the protein solutions on ice or adding protease inhibitors to the buffer.  

3. Freeze-Thaw – When the solution is frozen for storage and then thawed for use, the freeze-thawing causes multiple disturbances. It disrupts the natural conformation of protein. It also interferes with the precipitation and pH variation of buffer components. All of these factors contribute to loss of stability and protein denaturation. Adding glycerol to the buffer and storing in aliquots can help.  

4. Presence of heavy metals – Heavy metal salts are ionic. They interfere with salt bridges in protein and react with proteins resulting in insoluble metal protein salt. Including EDTA buffers reduces the denaturation of proteins by containing the heavy metals, preventing them from coming in contact with proteins. 

5. Oxidation – Proteins become unstable when they are oxidized. Adding strong reducing agents such as DTT in buffers can help prevent protein denaturation due to oxidation. 

6. Dilution Effects – Diluting the solution can affect protein stability.  Dilution induced denaturation can be prevented by maintaining protein concentrations at > 1mg/ml. 

7. Agitation – Shaking, vortexing, or stirring the solution vigorously can upset protein stability. Careful handling can help to maintain protein stability and prevent denaturation.