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 stable 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.
As mentioned above, the primary (and usually secondary) structure of proteins is quite rugged but tertiary structure can be disrupted quite easily. The probability of denaturing the protein increases with the number of manipulations required during processing. These manipulations include protein sample preparation, purification, and storage. Although different classes of proteins may require unique handling methods, the following rules of thumb apply to the handling of proteins for biochemical research in general.
The cardinal rule for handling proteins in the laboratory is to wear gloves. Wearing gloves in the chemical laboratory shields the researcher from harm, but in the biochemical laboratory, it’s the protein itself that requires protection. Besides preventing contamination from other laboratory reagents, the use of gloves prevents contamination from proteins commonly found on the skin, particularly proteases which may degrade the protein sample and destroy activity.
When developing conditions to stabilize a protein, it’s best to closely mimic the in vivo environment of the protein being handled. For example, protein solutions should be prepared in buffers at high concentration, preferably 1 mg/ml or greater. The high concentration tends to stabilize the protein’s native structure as well as inhibiting protein “sticking” to otherwise inert surfaces such as glass and plastic. If high concentrations of the native protein are unrealistic, addition of a second inert protein at high concentration will help prevent losses of protein on inert surfaces. Of course, the ability to easily separate the added protein from the sample protein is critical since addition of the secondary protein does constitute contamination of the sample. All glass and plasticware used for protein work should be judiciously washed with a good quality laboratory cleaner followed by thorough rinsing with deionized water. Rinsing with EDTA solution prior to deionized water reduces the chance of contamination by metal ions.
Proteins must be dissolved in buffers with great care. Vigorous shaking or stirring (e.g., vortex) can generate shear forces that in certain instances can destroy biological activity. Along with denaturation, protein solutions tend to foam uncontrollably when vortexed. A negative side effect here is oxygenation of the sample buffer, which can result in oxidation of the protein. Again, structure and biological activity can suffer in an oxygenated environment.
Storage of proteins for any length of time can pose stability problems. While working with proteins in the lab, they should be kept on ice. Since proteins are generally more stable at colder temperatures, maintenance at low temperatures even for short duration is recommended. Typically, proteins are stored freeze-dried (lyophilized), frozen in appropriate buffers, or refrigerated at 4°C. For short-term storage of proteins (hours to days), a standard laboratory refrigerator at 4°C 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 -20°C. 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 -20°C. 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, it’s advantageous to lyophilize a small aliquot to determine if the protein can be properly recovered.