Peptide Solubility
25/09/2021 2021-12-07 19:19Peptide Solubility
Peptide Solubility
What Factors Determine Peptide Solubility?
Occasionally, one of the more difficult aspects of conducting research with synthetic peptides can be determining the most effective solvent in which to dissolve the peptide. Many peptides dissolve easily in aqueous solutions (sterile water), but some researchers may encounter problems related to low solubility or even insolubility, particularly when working with peptides that contain long sequences of hydrophobic amino acids. However, researchers can predict any one peptide’s solubility by studying the known characteristics of its individual amino acids.
A peptide’s solubility is mainly determined by the physical properties of its amino acids. Amino acids can be classified as basic, acidic, polar uncharged, or non-polar. Non-polar amino acids are hydrophobic (they do not dissolve in aqueous solutions). Peptides containing a relatively large number of non-polar amino acids or polar uncharged amino acids generally dissolve more effectively in organic solvents such as DMSO, propanol, isopropanol, methanol, or DMF. Peptides with a high content of acidic amino acids can typically be dissolved in basic solvents (such as ammonium hydroxide, although this should not be used with peptides containing Cys), and, conversely, peptides containing a relatively high number of basic amino acids can generally be dissolved effectively in acidic solvents (such as acetic acid solution). However, researchers should always attempt to dissolve peptides in sterile water first, especially when the peptide contains less than five residues (amino acids), as these peptides generally dissolve quite easily in water.
Peptide Solubility Guidelines
Researchers should always test peptide solubility with a small amount of peptide in case ideal solubility is not initially achieved. Peptides should be allowed to warm to room temperature before attempting to dissolve them in solution. If attempts to dissolve the peptide in sterile water solution are unsuccessful, researchers should next try solutions that are able to be removed by lyophilization; if these solvents are not effective either, they can be removed by the lyophilization process, enabling the researcher to begin again without losing or compromising the peptide.
To assist with solubility, slight warming of solution (less than 40 Celsius or 104 Fahrenheit) or sonication techniques can be utilized. However, these techniques will only assist with dissolving; they will not alter a peptide’s inherent solubility characteristics. More information about peptide reconstitution can be found on our Peptide Reconstitution page.
Predicting Peptide Solubility Characteristics
To predict the solubility characteristics of a given peptide, the researcher must first evaluate the amino acid composition of the peptide, as the number and types of ionic charges in the peptide influence solubility. Specifically, it must be determined whether the peptide is acidic, basic, or neutral. To ascertain this, use the following steps:
1. Assign a value of -1 to acidic residues (amino acids). These include Asp (D), Glu (E), and C-terminal (COOH).
2. Assign a +1 value to each basic residue. These include Lys (K), Arg (R), and N-terminal NH2.
3. Assign a value of +1 to each His (H) residue at pH 6.
4. Calculate the overall net charge of the peptide by adding up the total number of the peptide’s charges.
Dissolving the Peptide in Solution
Once the overall net charge of the peptide has been calculated, solubility predictions can be made and the researcher can move on to dissolving the peptide in solution. Importantly, attempt to dissolve the peptide in sterile water solution first. If water is not effective, proceed to the following guidelines:
• If the overall net charge of the peptide is positive, attempt to dissolve the peptide in an acetic acid solution (10%-30%). If this is unsuccessful, try TFA (< 50 μl).
• If the peptide’s charge is negative, attempt to dissolve the peptide with ammonium hydroxide (NH4OH; < 50 μl). However, if the peptide contains Cys, do not use ammonium hydroxide; instead, add a small amount of DMF.
• If the peptide is neutral (overall net charge of 0), organic solvents are generally most effective. Attempt acetonitrile, methanol, or isopropanol. If the peptide is highly hydrophobic, attempt to dissolve it in a small amount of DMSO. Caution: peptides containing cysteine, methionine, or tryptophan are prone to oxidation by DMSO. Additionally, some peptides tend to aggregate (gel); for these peptides, add 6 M guanidine•HCl or 8 M urea.
Once the peptide has been successfully dissolved, dilute the peptide solution to the desired concentration by slowly adding the peptide solution into a buffered solution. Use gentle but constant agitation while combining to visually monitor and prevent localized concentration of the peptide in the aqueous solution. It is recommended to prepare the peptide stock solution at a higher concentration than is required by the experimental assay: the peptide stock solution can then be diluted further with the assay buffer.
Once the peptide solution has been prepared, it should be aliquoted as necessary and stored at -20C (-4F). For those peptides containing cysteine, methionine, or tryptophan, prevent oxidation damage by storing them in an oxygen-free environment.
Comment (1)
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