Solubility Guidelines for Peptides

Improper solubilization can potentially result in loss of the peptide/protein and result in failure of the experiment. When available, the solubility information may be found on the product information page or on the Certificate of Analysis. If the solubility information is not known, there are at least three fundamental requirements in selecting a solvent to dissolve the peptide or protein prior to use.

1. Select the solvent that effectively dissolves the protein.
2. The solvent has to be compatible with the experimental application.
3. The solvent should not react with or promote degradation of the protein.

Additionally, it is always a good idea to test the solubility of a small portion of the sample before dissolving the entire sample, and to choose an initial solvent that can be easily removed by lyophilization. This enables recovery of the peptide/protein from the solvent.

Determining Solubility Characteristics

Before adding any solvent to the lyophilized peptide, it is important to evaluate the amino acid composition of the peptide as a preliminary tool in understanding the solubility characteristics of your peptide. The number and types of ionic charges in the peptide determine its solubility in aqueous solutions. In general, the more charged residues the peptide possesses, the more soluble it is in aqueous solutions. In addition, peptides generally have more charges at pH 6–8 than at pH 2–6. It is for this reason that peptides are better dissolved at near neutral pH. Among the many exceptions to the rule are peptide sequences that are very hydrophobic and those that tend to aggregate. While the hydrophobicity of the sequence is the primary cause of aggregation, peptides can also aggregate or "gel" through extensive hydrogen bonding network. The guidelines below are used to determine if the peptide is basic, acidic or neutral.

1. Assign a value of -1 to each acidic residue (D, E, and C-terminal COOH).
2. Assign a value of +1 to each basic residue (K, R and the N-terminal NH₂).
3. Assign a value of +1 to each H residue at pH<6 and zero at pH >6.
4. Count the total number of charges of the peptide at pH 7 (all D, E, K, R, C-terminal COOH, and C-terminal NH₂).
5. Calculate the overall net charge of the peptide.

Dissolving Approach for Charged Peptides

Based on the above guidelines, proceed to test the solubility of the peptide using the following strategies:

1. If the overall net charge of the peptide is negative, the peptide is considered acidic. If the peptide is acidic, and/or if the total number of charges of the peptide at pH 7 is greater than 25% of the total number of residues, add a small amount of 0.1M ammonium bicarbonate to dissolve the peptide and dilute it with water to the desired concentration. Make certain that the resulting pH of the peptide solution is about 7 and adjust the pH as needed.
2. If the overall net charge of the peptide is positive, the peptide is considered basic. If the peptide is basic and the total number of charges of the peptide at pH 7 is between 10–25% of the total number of residues, add a small amount of 25% acetic acid to dissolve the peptide and dilute it with water to the desired concentration.
3. If the overall net charge of the peptide is zero, the peptide is considered neutral. If the total number of charges is greater than 25% of the total number of residues, use the strategy described in section 1. If the total number of charges is between 10–25% of the total number of residues, use organic solvents (see below).
4. If the total number of charges of the peptide is less than 10% of the total number of residues, the use of organic solvents is recommended.

Note: it is important to dissolve the peptide completely in the initial solvent (such as acetic acid, acetonitrile, DMSO or DMF) because the rate of dissolution of the peptides into these solvents is usually higher than in a water/solvent mixture. If the water/solvent mixture is used first to dissolve the peptide, you may end up adding a much larger than necessary amount of nonaqueous solvent to your peptide sample.

For any solvent used, the maximum concentration of the initial solvent will depend on the tolerance of your assay against that particular solvent. Before trying stronger solvents, it is necessary to sonicate the peptide solution to confirm that the peptide is insoluble in the solvent. Sonication enhances solubilization, breaking the solid peptide into smaller particles. If, after sonication, the solution has gelled, appears cloudy, or has visible particulates, the peptide has not dissolved completely but is suspended. At this point, a stronger solvent is necessary. If the peptide does not dissolve, lyophilize and remove the volatile buffer solution. Once the sample is dry, alternative solvents can be tried on the same sample.

After the peptide is dissolved in the initial solvent, especially those dissolved in organic solvents, dilute the peptide by slowly adding (dropwise) the peptide solution into the buffered solution with gentle but constant agitation. This is to prevent localized concentration of the peptide in the aqueous solution, which can potentially result in precipitation of the peptide. The added benefit of this strategy is that the possibility of precipitation can be visually monitored and acted upon accordingly.

Dissolving Approach for Hydrophobic/Uncharged Peptides

The above recommendations based on the charged nature of the peptide will likely be inadequate for dissolving peptides containing more than 50% hydrophobic residues in their sequence, neutral peptides with less than 25% charges, and/or peptides that has less than 10% charges. Under these conditions, the use of organic solvents is recommended, such as acetonitrile (ACN), dimethylsulfoxide (DMSO), or dimethylformamide (DMF).

Note: peptide sequences containing Cys (C) and Met (M) are unstable in DMSO.

Addition of chaotropic compounds such as guanidine hydrochloride or urea can facilitate in breaking up hydrophobic interactions or reduce the "gelling" of peptides by disrupting hydrogen bonding network. Again, the concentration of the initial organic solvent or chaotropic reagents will be dependent on the tolerance of your assay system.