The manufacturers sell peptides in freeze-dried powder or lyophilized form. Lyophilization is the technique whereby water gets removed from a solution upon freezing. The use of vacuum allows ice to be directly converted into vapor from solid without passing through the intermediate liquid phase. The powder either has a granular appearance or looks fluffy depending on the technique used for lyophilization.
The lyophilized peptides need to be dissolved in the solvent before use. There is no universal solvent that can be used for all the peptides. The choice of solvent is guided by peptide stability and compatibility of the specific solvent with bioassays. In this respect, sterile water or bacteriostatic water often is the first choice of solvent. In spite of being the first choice, sterile or bacteriostatic water cannot dissolve every peptide. Hence experimentation with different solvents is often required for optimizing the solvent of choice. Sodium chloride solution in water is not advised to be used for dissolving peptides so as to avoid precipitation with acetate salts.
The polarity or native charge of a peptide is the key determinant for the solvent of choice. Basic peptides require acidic solvents whereas acidic molecules require basic solvents for optimal dissolution. The hydrophobic and neutral molecules dissolve best in organic solvents like DMSO, propanol and acetic acid. The powder should be dissolved in a small volume of the organic solvent and further diluted in sterile water. Important to note is that peptides with methionine or free cysteine should not be d in DMSO to avoid side-chain oxidation else they are rendered inactive.
Peptides Reconstitution Guidelines
Selection of appropriate solvent demands use of sterile water, 0.1% acetic acid, or any solvent which can be easily removed by lyophilization. It is recommended to dissolve a small amount of the peptide in the chosen solvent before dissolving the complete vial. The molecule should always be dissolved to form a higher stock concentration than that required for the actual assays. It can be diluted further in the appropriate assay buffer to derive at the appropriate concentration for use in the bioassays.
Upon resuspension, sometimes visible particles persist in the peptide solution. The vial can then be sonicated to enhance the dissolution of the particles to yield a clear solution. Sonication does not modify the solubility of the peptide but expedites the breaking up of the particles to enhance solubility. Post sonication, the researcher has to inspect for the presence of any surface layer, cloudy appearance, or gel-like consistency. If so, then the peptide has merely got suspended but not dissolved. Hence, it would demand use of a stronger solvent.
Practical Implementation in the Laboratory
In this segment, we have outlined simple guidelines for the effective dissolution of peptides in the laboratory. Most peptides have recommended storage temperatures as lyophilized powder and solution and should be adhered to. If the powder form of the drug is stored in refrigerated condition, it should be equilibrated to room temperature initially before resuspension. The peptide solution can be filtered through a 0.2 µm filter to avoid any possible bacterial contamination since it is a rich source of amino acids.
Peptides Reconstitution – Example using sterile water as the diluent:
Step 1 – The plastic cap from the peptide vial should be removed so that the rubber stopper can be accessed.
Step 2 – The rubber stopper of the sterile water vial gets exposed upon removal of the plastic cover of the vial.
Step 3 – The rubber stoppers should be swabbed with alcohol to sterilize them from any possible bacterial contamination.
Step 4 – 2mL (milliliters) of water needs to aspirated from the sterile water vial.
Step 5 – 2mL (milliliters) of sterile water needs to be injected into the peptide vial, allowing the water to enter the vial gradually.
Step 6 – The vial should be gently swirled to allow the peptide to dissolve into the solvent. Shaking of the vial should be avoided.
Disclaimer: The products mentioned are not for human or animal consumption. All the information shared in this article is for educational purposes only.