Dissolution of peptides

Dissolving peptides is a very complex task, and it is generally difficult to determine the appropriate solvent at once.

Usually, a small amount is taken for testing first, and it is important not to dissolve everything until a suitable solvent is determined.

The main issue in peptide dissolution is the formation of secondary structures.  

Although the formation of the secondary structure of hydrophobic peptide chains is more pronounced, this phenomenon occurs in almost all peptide chains except for the shortest one, regardless of polarity. Therefore, the first principle for dissolving peptides is to use sterile distilled water or deionized water (or anaerobic water if conditions permit).  

Peptide solutions may encounter bacterial degradation. To avoid this phenomenon, peptides should be dissolved in sterile distilled water or filtered through 0.45 or 0.2 μ m pore size membranes for sterilization.  

Peptides containing Cys, Met, and Trp are particularly prone to oxidation and should therefore be dissolved in anaerobic water. Anaerobic water can be obtained by injecting inert gases (nitrogen, helium, argon) to reduce pressure and remove gas.  

If the peptide is insoluble in pure water, ultrasonic treatment can help break down the particles and increase solubility. Attention: Ultrasonic treatment can cause solution heating and peptide degradation.  

3. If the polypeptide contains multiple basic amino acids, use (1-10%) acetic acid aqueous solution; For peptides with very strong hydrophobicity, 50% acetic acid is used.  

If the polypeptide contains a large amount of acidic amino acids, it can be dissolved in ammonia solution (1-10%) or volatile alkaline buffer solutions such as ethyl morphine acetate or bicarbonate. The pH value must be adjusted before chromatography.  

5. Isopropanol and acetonitrile can dissolve medium-sized peptides. If the peptide is to be loaded onto the column, the amount of organic solvent must be very small, otherwise it will seriously affect the residence time.  

If the peptide is highly hydrophobic due to the presence of aromatic side chains such as Val, Leu, Met, Phe, Tyr, Ala, or is a neutral peptide, the use of membrane denaturing agents such as DMF or DMSO can help dissolve the peptide.  

a. High concentration membrane denaturing agents enhance solubility by disrupting the secondary structure of peptides.  

b. Membrane denaturing agents are suitable for the preparation of peptide analysis solutions, but may interfere with research on their biological activity.  

c. DMF is the optimal denaturing agent (with a maximum concentration of up to 30%), added dropwise until the peptide is dissolved.  

d. During reverse phase chromatography, DMF will flow out together with the eluent peak, and the peak may be high depending on the amount injected. Most peptides can flow out within a few minutes after a large amount of DMF is released. If the peptide chain is small and eluted too early, the amount of peptide will decrease.