What Factors Determine Peptide Solubility?
One of the more challenging aspects of conducting research with synthetic peptides is determining the most effective solvent. While many peptides dissolve easily in aqueous solutions (sterile water), researchers may encounter problems with peptides containing long sequences of hydrophobic amino acids. Understanding the characteristics of individual amino acids allows prediction of solubility behavior.
A peptide's solubility is primarily determined by the physical properties of its amino acids. These can be classified as:
- Basic amino acids: Lysine (K), Arginine (R), Histidine (H)
- Acidic amino acids: Aspartic acid (D), Glutamic acid (E)
- Polar uncharged: Serine, Threonine, Asparagine, Glutamine
- Non-polar (hydrophobic): Alanine, Valine, Leucine, Isoleucine, Phenylalanine, Tryptophan, Methionine, Proline
Solubility Guidelines
Peptides with high non-polar content generally dissolve better in organic solvents such as DMSO, propanol, isopropanol, methanol, or DMF. Peptides with high acidic content typically dissolve in basic solvents, while those with high basic content dissolve in acidic solvents.
Practical Recommendations
Before You Begin
- Test solubility with a small amount of peptide first
- Allow peptides to warm to room temperature before dissolving
- Try sterile water first, especially for peptides with fewer than five residues
To Assist Dissolution
- Gentle warming (below 40°C / 104°F)
- Sonication techniques
- These methods assist dissolution but don't change inherent solubility
Calculating Net Charge
To predict solubility, calculate the peptide's overall net charge:
- Assign -1 to acidic residues: Asp (D), Glu (E), and C-terminal COOH
- Assign +1 to basic residues: Lys (K), Arg (R), and N-terminal NH₂
- Assign +1 to His (H) residues at pH 6
- Sum all charges to determine net charge
Solvent Selection Based on Charge
Positive Net Charge
Try acetic acid solution (10-30%). If unsuccessful, try small amounts of TFA (<50 μL).
Negative Net Charge
Try ammonium hydroxide (NH₄OH, <50 μL). Note: Do not use ammonium hydroxide with peptides containing Cysteine—use small amounts of DMF instead.
Neutral (Net Charge = 0)
Organic solvents are generally most effective. Try acetonitrile, methanol, or isopropanol. For highly hydrophobic peptides, use small amounts of DMSO.
Caution: Peptides containing Cysteine, Methionine, or Tryptophan are prone to oxidation by DMSO. For peptides that aggregate, consider 6M guanidine·HCl or 8M urea.
Preparing the Final Solution
Once dissolved, dilute to the desired concentration by slowly adding the peptide solution to a buffered solution. Use gentle, constant agitation while combining. Prepare stock solutions at higher concentrations than needed—they can be further diluted with assay buffer.
Storage Recommendations
- Aliquot the solution as needed
- Store at -20°C (-4°F)
- For peptides containing Cys, Met, or Trp, store in an oxygen-free environment to prevent oxidation