Peptide Stability

    A common misconception about peptide is that peptide is as unstable as protein. In fact, peptide is much more stable than protein because the two factors affect protein stability do not exist for synthetic peptides. These two factors are the tertiary folding and proteinase contamination.

    A protein is prone to denaturation because its tertiary structure is held together by non-covalent bonds such as electrostatic interaction and hydrophobic interaction. Due to their short length, most peptides do not have sufficient amount of such interaction to fold each molecule into a defined tertiary structure. As a result, peptides can not be denatured as proteins do. Under this assumption, a peptide can only be damaged by covalent modification or break of peptide bonds. Unlike protein purified from cells that are full of various proteinase, the chance of protease contamination is extremely small for a synthetic peptide.

    Reactions could damage peptide such as oxidation require extreme pH. They are very slow under the neutral pH condition that most biological experiments are performed. Bacteria contamination is probably a more serious threaten than those reactions, because peptide is a good nutrition source for bacteria. Therefore, solvent filtration is important for peptide stability.

    In general, under normal conditions, peptide solution is stable for several days at room temperature, several weeks at 4 degrees and several months or more at -20 degrees. Dried peptide powder is stable for several months at room temperatures and several years at -20 degrees.

Peptide Concentration
    Accurate determination of peptide concentration is surprisingly more complicated than many expected. In fact, there is not a simple universal method for this purpose. Weighing and UV absorbency are two most common methods.
    a. Weighing. It can only provide a rough estimation for peptide amount. First, peptide powder is not easy to handle. The accuracy of weighing small amount (<3 mg) is especially questionable. Another problem is that peptides can contain various amount of water and to less extent, counter ions even after they have been extensively lyophilized. The content of water ranges from ~5% to 20%, some even >40%. The actual amount depends on peptide sequences. The types and amount of counter ions depends on solvents used for peptide purification and peptide sequence. The uncertainty generated by these water molecules cannot be ignored. 
    b. UV absorbency. If your peptide contains a Tyr or a Trp residue, peptide quantitation becomes much simpler. The molar absorbency [1/(M*cm)] of Trp at 282 nm is 5700 and that of Tyr at 275 nm is 1400. In 0.1N NaOH, the –OH group of Tyr is fully deprotonated. This shifts Tyr absorbency peak to 293 nm. Under this condition, its molar absorbency increases to 2400. One concern is that UV absorbency can be different if there are interactions between side chains in a peptide. This is not a problem for small hydrophilic peptides. In water solution, those peptides usually adopt an extended conformation in which all side chains are fully solvent exposed. But for long and hydrophobic peptides, this assumption is no longer a safe bet. Oligomerization and folding are sometimes observed. For this reason, we believe Tyr absorbency at 293 nm in 0.1 N NaOH is the best method because peptides are completely denatured under this condition. Its only drawback is that the sample used for concentration determination cannot be recovered.

    If your peptides do not contain a Trp or Tyr and accurate knowledge about peptide concentration is critical, the only reasonable option left is quantitative amino acid analysis. This is certainly beyond routine operation in most laboratories.

    Based on above discussion, we strongly suggest you to add a Tyr residue at either N- or C- terminal of your peptide when precise determination of peptide concentration is necessary.

 
The N-terminal Acetylation and the C-terminal Amide
    It is often thought that blocking the two ends with acetylation and amide respectively can increase stability of peptide. This is not true.

    The sequences of most synthetic peptides are originated from a segment of proteins. In a protein, both the N-terminal and the C-terminal ends of the peptide sequence form peptide bonds. This is different from the synthetic peptide that has charged free ends. The physical chemistry properties of a peptide can vary remarkably between the charged and uncharged forms, which in turn can affect the function of the peptide greatly. This problem can be resolved by blocking the N-terminal end with acetylation and the C-terminal end with amide, which makes both ends of a synthetic peptide more like peptide bonds. For this reason, the peptides we use for antibody production are end-blocked unless the antigen is located at the end of a protein. For the same reasons, we suggest customers to block both ends of the synthetic peptides used for other functional studies.

    There is no theoretical base to support that peptides with free ends are less stable.

Peptide Solubility
    Solubility becomes a concern only when a peptide is tried to be dissolved in aqueous solution, in which its biological function is performed. For organic solvent, solubility is not an issue at all ---- almost all peptides can be dissolved in organic solvents. But this won't help much because most studies about peptide can not be performed in organic solvents.

    The solubility of a peptide is ultimately determined by its sequence. If your peptide contains high percentage of hydrophobic residues, you are out of luck. Unfortunately, most researchers have to work with what they have unless they want to change their research projects. Among the conditions that can be manipulated, pH is probably the most important one. We found that in many cases, change of 1-3 of pH units could make a rock-solid peptide totally soluble. The transition curves are usually steep. Solution becomes crystal clear from cloud turbid within a narrow pH range. This is probably due to the change of ionization states of some residues, such as His.

    There is another reason to make pH adjustment important. Since HPLC solvents for peptide purification contain 0.1% TFA that cannot be completely removed by lyophilization, peptides being received often contain residual amount of TFA. This makes peptide solution often more acidic than you expect.

    For a peptide that is difficult to dissolve, you can always make a high concentration stock solution in an organic solvent, such as DMSO and dilute the peptide during biological function assay. Most assays can tolerate 1-2% DMSO, some even 5%. This does not always work, however. Some peptides aggregate or precipitate after they are diluted out from DMSO even at low concentration.