The solubility of peptides depends on polarity

The solubility of a peptide depends largely on the polarity of the peptide. Acidic proteins are dissolved in alkaline solutions, while alkaline proteins can be dissolved in acidic solutions. Hydrophobic and neutral peptides containing large amounts of uncharged polar amino acid residues or hydrophobic amino acids can be dissolved in a small amount of organic solvents, such as DMSO, DMF, acetic acid, acetonitrile, methanol, propyl alcohol or isopropyl alcohol, and then diluted with water (distilled water). Peptides containing methionine or cysteine cannot be dissolved by DMSO because DMSO may cause side chain oxidation.

Peptide dissolution test

Before the peptide is dissolved, take a small portion for a peptide dissolution test. You need to test several different solvents until you find the most appropriate one. Ultrasonic treatment helps to break up particles and increase solubility. (Note: Ultrasonic treatment can cause solution heating and peptide degradation.)

1: Assign a value of -1 to each acidic amino acid, including aspartic acid (D), glutamic acid (E), and carboxy-terminal -COOH. Each basic amino acid is assigned a value of +1, including arginine (R), lysine (K), histidine (H), and the amino terminus -NH2. And then calculate the charge of the entire peptide.

2: If the charge of the entire peptide is positive, the peptide is basic. Try dissolving with distilled water first; If it does not dissolve in water, then try to dissolve with a small amount of 10%-25% acetic acid. If that still fails, add some TFA (10-50 µl) to increase the solubility, then dilute with water to the ideal concentration.

3: If the charge of the entire peptide is negative, the peptide is acidic. Acidic peptides can be dissolved with PBS (PH 7.4). If not, add a small amount of alkaline solvent, such as 0.1 M ammonium bicarbonate, and then dilute with water to the ideal concentration. peptides containing free cysteine should be dissolved in degassed acid buffers because the sulfhydryl groups are rapidly oxidized to disulfide at PH greater than 7.

4: If the entire peptide charge is zero, the peptide is neutral. Neutral peptides are usually dissolved in organic solvents. First, try adding a small amount of acetonitrile, methanol, or isopropyl alcohol. For highly hydrophobic peptides, a small amount of dimethyl sulfoxide can be dissolved and then diluted with water to an ideal concentration. For peptides containing free cysteine, DMF should be used instead of DMSO. For peptides with a tendency to aggregate, 6M guanidine hydrochloride or 8M urea can be added and diluted as necessary.

The storage of peptides

To prevent or minimize peptide degradation, store the peptides in freeze-dried powder at -20°C, or preferably -80°C. If solution peptides need to be preserved, it is best to store them in small samples to avoid repeated freeze-thaw. If a sample is not used up after thawing and freezing, it should be thrown away. Bacterial degradation can sometimes be a problem for solution peptides, so dissolve the peptide in sterile water or peptide solution and filter to remove bacteria.

Basic amino acids: K, R, H, N-terminus

Acid amino acids: D, E, C-terminus

Polar neutral amino acids: F, I, L, M, V, W, Y

Non-polar hydrophobic amino acids: G, A, S, T, C, N, Q, P, acetyl, amide

For example:

RKDEFILGASRHD: (+5) + (-4) = +1 is considered to be an alkaline polypeptide, see Step 2

EKDEFILGASEHR: (+4) + (-5) = -1 is considered to be an acidic polypeptide, see Step 3

AKDEFILGASEHR: (+4) + (-4) = 0 is considered a neutral peptide, see Step 4