Académique Documents
Professionnel Documents
Culture Documents
The maintenance of protein stability through every phase of laboratory research, such as homogenization and freeze-drying, is critical for success. Proteins are large biological polymers made up of approximately twenty naturally occurring amino acids. The primary structure, that is the sequence of amino acids composing the backbone of the protein, is held together by peptide bonds. These peptide bonds correspond to a class of organic compounds called amides and are generally stabile under typical (and not so typical) biological conditions. Secondary and tertiary structures represent the folding of the protein onto itself as a result of the nature of the side chains of the constituent amino acids. Secondary and tertiary structure can be disrupted quite easily, especially during routine handling in vitro. Since a denatured protein corresponding to disruption of tertiary structure is generally useless, the goal of any process involving the use of active proteins must include scrupulous maintenance of protein stability and biological function.
refrigerator at 4C is satisfactory providing the buffer used to solvate the protein provides all the necessary components necessary to stabilize the protein of interest. These components can include reducing agents, hydrophobic additives, and protease inhibitors added to buffers. Along with the use of gloves mentioned previously, protease inhibitors prevent denaturation due to contamination from these lytic agents potentially present in the protein source. Additionally, antibacterial agents such as sodium azide can be added to inhibit bacterial growth. Care must be taken, however, since antibacterial agents and protease inhibitors represent deliberate contamination of the sample. Proper controls must be evaluated to insure no deleterious interaction with the protein of interest will occur.Proteins can be stored long term (days to weeks) by quick-freezing the sample followed by storage at -20C. Addition of stabilizers such as glycerol helps prevent damage to the protein during freezing and thawing. Typical concentrations for glycerol are 10% to 50%. Again, care must be exercised since glycerol may negatively effect any chromatography methods subsequently used for sample handling or further purification after thawing of the sample. Although stable while frozen, repeated thawing and freezing of a sample can lead to degradation and loss of activity. During the freezing process proteins are exposed to extremes of salt concentration and pH. Along with the use of stabilizers such as glycerol, rapid freezing of the protein solution limits the time the protein is exposed to these extreme conditions. The rapid freezing process is typically performed by immersing the protein solution in a dry ice bath containing either acetone or ethanol followed by frozen storage at -20C. Along with rapid freezing, the thawing process should also be rapid for the same rationale as when freezing. This can be accomplished by immersion in running lukewarm water. Even when performed rapidly, repeated freezing and thawing of protein samples is considered ill advised. It is advised to divide the original protein sample into several individual aliquots. As sample is needed, a lone aliquot is thawed. In this way the entire sample is not exposed to the perils of repeated phase changes. Finally, lyophilization can also be used for long-term protein storage. Here the protein will eventually be reduced to a dehydrated powder for convenient storage in a laboratory freezer. Although theoretically ideal, there are several hazards along the way. As before, the protein must be rapidly frozen to avoid the pitfalls previously mentioned. The protein must be dissolved in either deionized water or buffer containing lyophilizable salts. If not, buffer salts will remain with the protein after the lyophilization process is complete. After the protein solution is frozen, it is attached to a lyophilizer where the frozen solution sublimes leaving the protein behind, usually as a fluffy white powder. A major problem that occurs quite frequently with lyophilization is the inability to redissolve the lyophilized protein, which indicates denaturation during the process. Prior to lyophilizing the entire protein sample, its advantageous to lyophilize a small aliquot to determine if the protein can be properly recovered.
The interactions between proteins and solvent components have been investigated for the sucrose/water system. Thermodynamic and kinetic measurements of the thermal unfolding of alpha-chymotrypsin, chymotrypsinogen, and ribonuclease were performed as a function of sucrose concentration. The alteration in protein-solvent interactions in the presence of sucrose was also studied by density measurements and analyzed by multicomponent thermodynamic theory. Sucrose does not induce a conformational change in three proteins studied, although it does induce a small change in the circular dichroism spectrum of ribonuclease. The enthalpy of thermal unfolding shows little dependence on the concentration of sucrose, while the apparent activation energy of the unfolding process is increased by the addition of sucrose. The results from the protein-solvent interaction study indicate that sucrose is preferentially excluded from the protein domain, increasing the free energy of the system. Thermodynamically this leads to protein stabilization since the unfolded state of the protein becomes thermodynamically even less favorable in the presence of sucrose. The exclusion of sucrose from the protein domain seems to be related to the higher cohesive force of the sucrose water solvent system since all the experimental observations can be correlated with the effect of sucrose on the surface
tension of water