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HANDLING OF PEPTIDES

Frequently Asked Questions on Handling of Peptides

How to handle a peptide lyophilizate?

Peptides, especially short ones and arginine-rich peptides, can be very hygroscopic. Hence they have to be protected from humidity and handled rapidly.

Peptides should not be subjected to strong light, extreme pH-values, and oxidants.

As peptides are biologically active even in minute amounts, a dust mask, safety goggles, and protective gloves should be worn when handling lyophilizates.

How to weigh out a peptide?

Peptides usually are provided as lyophilizates, which tend to be hygroscopic. Adsorption of humidity will reduce the peptide content and adhered water may decrease stability. Before opening, the vial should be allowed to reach ambient temperature in a desiccator. Lyophilizates often “stick” due to electrostatic charging. The peptide has to be weighed out quickly and the vial resealed.

Except for handling very short peptides or large amounts, reconstituting the peptide and aliquoting the resulting solution is preferable to weighing out.

How to dissolve a peptide?

Please see our Technical Note “Solubilization of Peptides”, which can be downloaded from our website.
The solubility of a peptide in a solvent or buffer is determined mainly by its polarity, which results from the amino acid composition, and its conformation.
Solubility in water can be expected for peptides containing a large proportion of charged amino acids. As the peptides are usually produced as trifluoroacetate salts, the basic amino acids (Arg, Lys, His) and the N-terminal amino group will be protonated. The aqueous solubility of peptides containing Asp and/or Glu can be improved by adding diluted ammonia, which will generate the ammonium salts.
Polar amino acids as Asn, Gln, Gly, Ser, Thr facilitate reconstitution in water.
DMSO (dimethyl sulfoxide) is a highly suitable solvent for dissolving non-polar peptides (peptides containing a large proportion of Ile, Leu, Met, Phe, Pro, Trp, Val; most fluorophoric and chromophoric peptide substrates), it will also disrupt aggregates. Addition of DMSO (or DMF, acetonitrile, or other highly polar water-miscible solvents) may help to reconstitute a peptide, which is scarcely soluble in water. DMSO should not be used in combination with strong acids as trifluoroacetic acid. Most peptides will dissolve in acetic acid. Cys- and Met-containing peptides require special attention to prevent oxidation of the sulfur.
Reconstitution of peptides, especially long peptides or inner salts, may take time. Gentle warming or short repetitive sonication to accelerate dissolution is usually tolerated.
The intended use may limit the choice of solvents.
We advise against dissolving peptides directly in assay buffer, except if they show a high water-solubility.

Can I predict if a peptide is soluble in aqueous buffer?

Unfortunately, the solubility of a peptide in water cannot be predicted just by studying the structure. However, a few clues can be deduced from the sequence: a relatively short peptide containing Lys and Arg residues will be soluble in aqueous buffers, as most basic functionalities will be protonated in peptides sold as trifluoroacetate salts. The guanidine function of Arg is a strong base, whereas the ε-amino group of Lys is a moderately strong base. By contrast, “acidic” peptides containing a large proportion of Asp and Glu tend to be insoluble in water, but they are readily dissolved by diluted ammonia, and by basic buffers. The side-chain carboxy functions are rather weak acids, they are considerably less acidic than the C-terminal carboxyl group.
Degassed buffers have to be used for reconstituting Cys- and Met-containing peptides. Contrary to sulfoxide formation, the oxidation of the thiol moiety is pH-dependent. Peptides containing free cysteines should be dissolved in acidic buffers.

How to dissolve peptides containing free cysteines?

Peptides containing a single free cysteine will be oxidized at pH>7 yielding dimers. This oxidation can be reverted. Peptides containing two or more thiol moieties yield a mixture of products upon oxidation. pH 7.5-8 is the pH optimum for disulfide bond formation. Hence, peptides containing free cysteines are best dissolved in degassed solvents, e.g. buffers pH<7, diluted acetic acid, 0.1% trifluoroacetic acid in aqueous acetonitrile.

DMSO should be avoided, especially with peptide trifluoroacetates.

How to dissolve peptides containing disulfide bridges?

Basic buffers should be avoided.

How to dissolve β-amyloid fragments (up to Aβ 1-40)?

β-Amyloid peptides will form insoluble aggregates during storage.
“Fresh” lyophilizates of Aβ (25-35) and (1-40) are still soluble in water (oxygen-free water has to be used), the “aged” peptides may require the addition of acetic acid for dissolution.
Direct dissolution of amyloid peptides in buffers as PBS should not be attempted, better dissolve them first in water or 50% acetic acid and then dilute with the working buffer.
Please see also our Technical Note “Care and Handling of Amyloid Peptides”, which can be downloaded from our website.

How to dissolve amyloid β protein (1-42) (Aβ 1-42)?

Aβ (1-42), its mutants, and longer amyloid fragments will form insoluble aggregates during storage.  Aβ (1-42) is soluble in hexafluoroisopropanol (HFIP), DMSO, or bases as 0.1% aqueous ammonia, 50 mM TRIS ∙ HCl, or 1mM NaOH, which all revert aggregation. Reconstitution in HFIP or DMSO takes time whereas ammonia rapidly dissolves the peptide.
Aβ (1-42) solutions in DMSO or aqueous bases can be diluted directly with working buffer. The volatile solvent HFIP is usually evaporated leaving a residue of monomeric, soluble Aβ (1-42), which can be reconstituted with the chosen buffer pH 7.4 as to induce fibrillation.

How should peptides be stored?

For long-term storage the peptide should be kept in solid form in the deep freezer at < -15 °C. For short-time storage a refrigerator (+4 °C) will suffice.

Peptides should be protected from intense sunlight.

 

Peptides containing fluorophores should be kept in the dark.

How long can I store peptides in solid form (as lyophilizate)?

The shelf stability of peptides is sequence-dependent. A few amino acids and partial sequences may react, even at low temperatures, e.g. Cys, Met, and Trp can be oxidized, -Asn-Gly- and -Asn-Ser- form aspartimide yielding further cleavage products.

Lyophilizates contain water, which may be involved in degradation reactions during storage. Peptides containing Asn, Gln, Cys, Met, Trp tend to be less stable. Moreover, the salt form affects stability.

The stability may range from few months to years, if the peptide is kept in a tightly closeed vessel at <-15°C.

How long can I store peptide solutions?

Dissolved peptides are less stable than the lyophilizates. The solutions are best aliquoted before freezing to avoid thawing-refreezing cycles stressing the peptide. Solution stability depends on the type of solvent and the pH (pH 5-7 is considered as optimum). Peptides containing Asn, Gln, Cys, Met, Trp, Tyr tend to be less stable. Stock solutions of peptide substrates are best prepared in dry organic solvents to avoid premature hydrolysis.

The stability of the aliquots may range from weeks to months.

What may happen during storage?

Typical degradation reactions are:
Oxidation of Met yielding the sulfoxide
Oxidation of Trp and Tyr
Oxidation of Cys to cysteic acid
Deamidation of Asn, Gln, and the C-terminal amide
Aspartimide formation
Cleavage of Asn-Pro
Pyroglutamine formation of N-terminal Gln
(Cyclization of N-terminal Glu has only rarely been observed)
Dimerization of Trp and Tyr
Racemization

The peptide I’ve dissolved is rapidly losing its activity. Why? Can I prevent it?

There could be many reasons for loss of activity, though oxidation of Met yielding the sulfoxide is a common one. The rate of sulfoxide formation is sequence-dependent. The problem can be circumvented by replacing Met with its stable isostere Nle. Bachem offers Nle analogs of a number of Met-containing peptides. If the Nle peptide you require is not available, please ask for a quote.
Sulfotyrosine-containing peptides may loose their activity due to desulfation, Gla-containg peptides can decarboxylate.

Why do you recommend the solvent mentioned on the ADS?

The mentioned solvent has been used for analyzing the peptide.
Only solvents, which rapidly dissolve the peptide in the required concentration without damaging the compound, are suitable for analytical purposes. Volatility or high UV-transmission can be additional criteria. This excludes e.g. the use of DMSO.

What is the difference between Aβ 1-42 H-1368 and H-6466?

H-1368 and H-6466 do not differ in solubility, but the hydrochloride fibrillates more readily than the inner salt H-1368.