How To Find The Best Peptide Solubility Option

Figuring out the most effective solvent to dissolve peptides with is possibly one of the most difficult components when working with peptides and conducting research. Aqueous solutions–also known as sterile waters–are one way to dissolve peptides. Problems do, however, still arise with this method. Some issues you may encounter are related to low solubility or even solubility. This matter is more common when working with peptides containing long hydrophobic amino acid sequences. Though there are difficulties, in this day and age, researchers can predict a peptide’s solubility just by studying its known characteristics and its amino acid.

An Amino Acid’s physical properties are what predominantly determines a peptide’s solubility. Amino Acids classification can be any one of the following four:
1. Basic
2. Acidic
3. Polar uncharged
4. Non-polar (hydrophobic-do not dissolve in aqueous solutions)

A large number of non-polar or polar uncharged amino acids dissolve more effectively with organic solvents such as:
2. Propanol
3. Isopropanol
4. Methanol
5. DMF

Basic solvents (ammonium hydroxide) are of better use for peptides with high content amino acids. It is important to note that ammonium hydroxide should not be used with peptides having Cys. Acidic solvents, such as acetic acid solution, are also better paired with peptides that contain a high number of basic amino acids. Each peptide dissolves more easily and effectively when paired with the correct solvent. The first step to take into account is that researchers should first attempt to dissolve peptides in sterile water. This is important, especially if a peptide has less than 5 amino acids since they dissolve more easily in water.

Steps To Remember For Proper Peptide Solubility

1. In order to achieve the ideal solubility, researchers must test a small amount of peptide in peptide solubility. This helps the researcher decipher whether the solution is a good match with the peptide.

2. Allow time for the peptide to reach room temperature before attempting to dissolve it in solution.
*Utilize solutions that are able to be removed by lyophilization if sterile water solution does not work when trying to dissolve the peptide. If none of the options are successful, then it can be removed by the lyophilization which allows the researcher to start the process again without losing or affecting the peptide.

3. A slightly warmer solution, for example less than 40 Celsius or 104 Fahrenheit, would aid in solubility. Sonication techniques can also be of use. It is vital to note that this only helps in dissolving a peptide. It will not change the peptides natural characteristics.

Researchers should evaluate an amino acids composition of the peptide to predict the solubility characteristics of a peptide. It is essential to check the number and type of ionic charges of a peptide influence solubility. It is imperative as it helps tell whether the peptide is acidic, basic, or neutral. To figure this out, one would assign a value of -1 to amino acids (also known as residues). For example, you may see Asp (D), Glu (E), and C-terminal (COOH). Then you would assign +1 value to each basic amino acid which includes Lys (K), Arg (R), and N-terminal NH2. You would also assign value to +1 to each His (H) amino acid at pH6. And finally calculate the net charge overall of the peptide. This is done by adding up the peptide’s total number.

What To Do When It Is Time To Conduct Peptide Solubility

Only when the net charge of the peptide is calculated can solubility predictions be made. This allows researchers to begin dissolving the peptides in solution. As mentioned previously it is also essential to try to dissolve peptides in sterile water before attempting to use other solutions. If and only when the water is ineffective, researchers may follow these directions.

First, you attempt to dissolve a peptide in acetic acid solution (10-30%) only if the overall net charge is positive. If this is not the case, try TFA (<50μl). Alternatively, if there is a negatively charged peptide, you may experiment with using ammonium hydroxide (NH4OH; < 50 μl) to dissolve the peptide. Note that if Cys is in the peptide, add a small amount of DMF and do not work with ammonium hydroxide. Lastly, If the overall net charge is 0, meaning the peptide is neutral, the most effective solvent would be organic solvents. Organic solvents consist of acetonitrile, methanol, or isopropanol. Use a small amount of DMSO if a peptide is highly hydrophobic.

CAUTION: DMSO can oxidize peptides containing cysteine, methionine, or tryptophan. Some peptides also tend to aggregate (gel); in regard to these peptides, add 6 M guanidine•HCl or 8 M urea.

Dilute the peptide solution to the desired concentrate by slowly adding the peptide solution into a buffered solution only when the peptide has successfully dissolved. It is critical to use gentle but, most importantly, constant agitation while combining it to monitor and avoid localized concentration of the peptide in an aqueous solution. The experimental assay also suggests preparing peptide stock solution at higher concentrations than what is typically required. This can help dilute the stock more with use of the assay buffer.

When the process is finished and the solution is prepared correctly, it should be moved and stored at -20C (-4F). Peptides that contain cysteine, methionine, or tryptophan should be stored
In an oxygen-free environment to prevent oxidative damage.

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