Proteins are often fragile molecules that need to be protected during purification and characterization. Protein denaturation refers to the loss of protein structure due to unfolding. Maintaining biological
activity is often important and protein denaturation should be avoided in those situations. Elevated temperatures, extremes in pH, and changes in chemical or physical environment can all lead to protein denaturation. In general, things that destabilize H-bonding and other forces that contribute to secondary and tertiary protein structure will promote protein denaturation. Different proteins exhibit different degrees of sensitivity to denaturing agents and some proteins can be re-folded to their correct conformations following denaturation.
Factors Affecting Protein Stability
· Temperature :
Increase in temperature can disrupt the hydrogen bonds and other non-polar hydrophobic interactions.
The reason behind this is increased temperature increases the kinetic energy and causes the molecules to vibrate so rapidly and violently that the bonds are disrupted.
One of the reason behind the use of heat for sterilization is this, because higher temperature leads to the denaturation of proteins of bacetrial cells there by killing the bacteria.
Example: Protein Coagulation and re-association of egg-white on frying an egg.
Avoiding high temperatures and Keeping protein solutions on ice can reduce temperature induced denaturation.
· Freeze-thaw :
Freezing and thawing can disturb the native conformation of proteins, pH variation and precipation of buffer components all those can lead to denaturation of proteins.
To avoid freezing induced denaturation determine effects of freezing. Include glycerol in buffers. Store in aliquots.so it will be easy to take out an aliquot and work with instead of freeze thawing the entire solution.
· Physical denaturation:
Physical denaturation occur due to handling of proteins, shaking induces aggregation of proteins, vortexing shaking etc can denature proteins very easily
Do not shake, vortex or stir vigorously. (Protein solutions should not foam.), which helps in reducing protein denaturation.
· Solution effects: To avoid protein denaturation due to the effect of solution, mimicking the cellular environment: maintaining neutral pH, ionic composition, etc.
· Dilution effects: dilution effects can be overcome by maintaining protein concentrations > 1 mg/ml as much as possible.
Oxidation of protein can destroy the stability of proteins, to prevent oxidation induced denaturation strong reducing agents such as DTT (or β-ME) in buffers can added.
· Heavy metals :
Heavy metal salts usually contains Hg2+, Pb2+, Ag2+, Ti1+, Cd2+ and other metalls with weigh atomic weights. Since salts are ionic they disrupt salt bridges in proteins, The reaction of heavy metal salts with protein usually leads to an insoluble metal protein salt.
Including EDTA in buffers helps in sequestering the heavy metals there by reducing protein denaturation.
· Microbial growth:
To prevent microbial growth sterile solutions can be used or including anti-microbials, and/or freezing helps to prevent microbial growth.
Proteases cleaves the protein and thus stability of the protein will be lost, resulting in denatured proteins, Include protease inhibitors. Keeping protein solutions on ice helps reduce the effect of proteases.
The optimal conditions for maintaining the stability of each individual protein need to be determined empirically. In general, though, protein solutions should be kept cold (< 4o C) except during assays and other procedures requiring specific temperatures. Many proteins are especially labile and need to be stored at -20oC or -80oC. However, repeated freezing and thawing of protein solutions is often deleterious. Adding 50% glycerol to storage buffers will lower the freezing point and allow storage at -20o. Solutions for working with proteins will often contain heavy-metal chelators and/or antioxidants as protectants.. In addition, proteases may be released during cell disruption and it may therefore be necessary to include protease inhibitors.