What is Counter-ion?
Also called: Counterion, Salt form
An ion of opposite charge that balances a peptide's charged groups in the solid state; in synthetic peptides it is usually trifluoroacetate, acetate or chloride.
By the APL Research Team · Updated
A peptide purified and dried from acidic solution is a salt. Its protonated amines carry positive charges, and each is balanced by an anion from the last acid the peptide met: trifluoroacetate after standard synthesis and purification, acetate or chloride after exchange. That anion is the counter-ion, and it is weighed along with the peptide every time powder goes on a balance. A review of impurities in peptide medicines treats unwanted counter-ions, trifluoroacetate in particular, as one of the impurity classes that can carry into a final product [1].
Counting binding sites
Under the acidic conditions of cleavage and purification, these groups are protonated and each pairs with one counter-ion:
| Group | Binds a counter-ion? |
|---|---|
| Free N-terminal amine | Yes; not if the N-terminus is acetylated |
| Lysine side chain | Yes |
| Arginine side chain | Yes |
| Histidine side chain | Usually; the imidazole is protonated at low pH |
| Asp, Glu and C-terminal carboxyl groups | No; largely uncharged at low pH |
For a fully stoichiometric salt, formula weight = peptide MW + n × (MW of the acid), because each site gains both a proton and an anion. The acids weigh 114.02 g/mol (TFA), 60.05 g/mol (acetic acid) and 36.46 g/mol (HCl).
Worked example: three peptides, three salt forms
| Peptide | Basic sites | Free peptide (g/mol) | Peptide fraction as TFA salt | As acetate | As hydrochloride |
|---|---|---|---|---|---|
| BPC-157 (GEPPPGKPADDAGLV) | 2: N-terminus, Lys | 1419.54 | 86.2% | 92.2% | 95.1% |
| Selank (TKPRPGP) | 3: N-terminus, Lys, Arg | 751.87 | 68.7% | 80.7% | 87.3% |
| MOTS-c (MRWQEMGYIFYPRKLR) | 5: N-terminus, Lys, 3 × Arg | 2174.59 | 79.2% | 87.9% | 92.3% |
Fractions exclude water. Two patterns stand out. Short peptides with many basic residues lose the most mass to counter-ions, and switching from trifluoroacetate to acetate moves the peptide fraction by 6 to 12 percentage points for these three sequences, a larger effect on stock concentration than the difference between any two common purity grades.
Why it matters at the bench
- Concentration. Weighing a salt and dividing by the free-peptide MW overstates molar concentration by the inverse of the peptide fraction: 1/0.687, or about 1.46-fold, for Selank as its trifluoroacetate. Net peptide content and the molarity calculator handle the correction.
- Biology. The counter-ion can change the result. Acetate, hydrochloride and trifluoroacetate salts of five antimicrobial peptides differed substantially in antistaphylococcal activity, haemolysis and keratinocyte cytotoxicity, and no single salt was best across all five [2]. Trifluoroacetate has the largest literature of its own.
- pH and solubility. In unbuffered water, the counter-ion and the peptide's ionisable groups set the pH, which in turn affects how far the solution sits from the peptide's isoelectric point.
- Real content differs from theory. Exchange is often partial and excess acid can remain, so actual counter-ion content is measured; ¹⁹F-NMR is one way to follow residual trifluoroacetate [3].
The acetate salt entry covers exchange, and the TFA versus acetate guide when the salt form is worth specifying.
References
- 1.D'Hondt M, Bracke N, Taevernier L, et al. Related impurities in peptide medicines. J Pharm Biomed Anal. 2014. PubMed 25044089
- 2.Sikora K, Jaśkiewicz M, Neubauer D, et al. Counter-ion effect on antistaphylococcal activity and cytotoxicity of selected antimicrobial peptides. Amino Acids. 2018. PubMed 29307075
- 3.Roux S, Zékri E, Rousseau B, et al. Elimination and exchange of trifluoroacetate counter-ion from cationic peptides: a critical evaluation of different approaches. J Pept Sci. 2008. PubMed 18035848