A sequence tells you more than a mass. From the composition alone you can estimate how a peptide will behave in solution: its charge at the pH of your buffer, the pH at which it is least soluble, how hydrophobic it is, and whether you can measure its concentration by UV absorbance.
Net charge at a chosen pH
Ionisable groups gain or lose a proton depending on pH. Each group’s average charge follows the Henderson–Hasselbalch relationship:
basic group: +1 / (1 + 10pH − pKa) · acidic group: −1 / (1 + 10pKa − pH)
The net charge is the sum over the free N-terminus and Lys, Arg and His (basic), plus the free C-terminus and Asp, Glu, Cys and Tyr (acidic). Capped termini (acetylated or amidated) carry no charge, and cysteines locked in disulfide bonds cannot ionise.
| Group | pKa used | Group | pKa used |
|---|---|---|---|
| N-terminus | 8.6 |
C-terminus | 3.6 |
| Lys (K) | 10.8 |
Asp (D) | 3.9 |
| Arg (R) | 12.5 |
Glu (E) | 4.1 |
| His (H) | 6.5 |
Cys (C) | 8.5 |
| Tyr (Y) | 10.1 |
These are the EMBOSS values. Other tools use slightly different sets, which is why two calculators can disagree by a few tenths of a pH unit.
Isoelectric point (pI)
The pI is the pH at which the net charge is zero. Peptides are often least soluble near their pI, because there is no net charge to keep molecules apart. The calculator finds the pI by bisection: it evaluates the charge across pH 0–14 and narrows in on the zero crossing.
GRAVY: grand average of hydropathy
Each amino acid has a Kyte–Doolittle hydropathy value, from +4.5 for isoleucine to −4.5 for arginine. GRAVY is their average over the sequence. Positive scores suggest a hydrophobic peptide that may need an organic co-solvent. Negative scores suggest a hydrophilic peptide that should dissolve in aqueous buffer. GRAVY is a coarse guide: two peptides with the same score can behave quite differently depending on where the hydrophobic residues sit.
Extinction coefficient at 280 nm
At 280 nm, absorbance comes almost entirely from tryptophan, tyrosine and disulfide-bonded cystines. Pace and colleagues give the standard estimate:
ε280 = 5500·nTrp + 1490·nTyr + 125·ncystine (M−1cm−1)
With ε you can find concentration from a UV reading using Beer–Lambert, c = A / (ε × l). The analyzer also reports the absorbance of a 1 mg/mL solution (ε ÷ MW), which is handy for spectrophotometers that work in mass units.
Worked example: YGGFL (Leu-enkephalin)
One Tyr and no Trp or cystines: ε280 = 1490 M−1cm−1. With a molecular weight of 555.63 g/mol, a 1 mg/mL solution reads about A280 = 1490 ÷ 555.63 ≈ 2.68 in a 1 cm cuvette.
When A280 does not work
- No Trp or Tyr. Many short peptides, GHK for example, have ε280 = 0 and cannot be quantified this way. Use weighing with net peptide content, amino acid analysis or absorbance at 205–214 nm instead.
- Low ε. With a single tyrosine, dilute solutions give readings near the noise floor of the instrument.
- Interfering buffer components. Some additives absorb in the UV, so blank against the same buffer.
References
- EMBOSS iep documentation
- Grimsley GR, Scholtz JM, Pace CN (2009). A summary of the measured pK values of the ionizable groups in folded proteins. Protein Sci 18:247–251
- Kyte J, Doolittle RF (1982). A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132
- Pace CN, Vajdos F, Fee L, Grimsley G, Gray T (1995). How to measure and predict the molar absorption coefficient of a protein. Protein Sci 4:2411–2423
- Gill SC, von Hippel PH (1989). Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182:319–326
- ExPASy ProtParam documentation