What each property tells you
Molecular weight sets the conversion between mass and moles. Isoelectric point tells you the pH at which the peptide is least soluble and most likely to precipitate. Net charge at pH 7 tells you how it will behave on an ion exchange column and how strongly it interacts with charged surfaces.
GRAVY summarises the overall hydrophobicity of the sequence, which predicts whether water alone will dissolve it. The extinction coefficient tells you whether you can quantify it by ultraviolet absorbance at all. The aliphatic index is a documented correlate of thermostability.
Isoelectric point is a solubility warning
At its pI a peptide carries no net charge, so the electrostatic repulsion that keeps molecules apart in solution disappears and aggregation becomes much more likely. Solubility is at its minimum there.
The practical consequence is to keep your buffer at least one pH unit away from the pI. A peptide with a pI of 7.2 in a phosphate buffer at pH 7.4 is being handled at exactly the worst point on its solubility curve.
Net charge predicts surface behaviour
A strongly cationic peptide at physiological pH binds to negatively charged surfaces: glass, some plastics, and cell membranes. This shows up as unexplained loss during handling, and it is why cationic antimicrobial peptides are usually handled in low-binding plasticware.
It also determines which ion exchange chemistry will retain the peptide, which matters if you are purifying rather than buying.
Reading GRAVY correctly
GRAVY is the mean Kyte-Doolittle hydropathy value across the sequence. Positive means hydrophobic on average, negative means hydrophilic. Values below about minus one are reliably water-soluble; values above about plus 0.5 usually need a co-solvent.
Being an average, it hides distribution. A peptide with a strongly hydrophobic stretch and a strongly hydrophilic one can average close to zero while behaving like neither. The hydrophobicity plotter shows the profile along the sequence, which is the view that catches this.
Why the aliphatic index replaced the instability index here
This calculator used to display a figure labelled instability index, calculated as the percentage of aspartate, proline, serine, threonine and glycine residues, with a threshold at 40 borrowed from the real statistic. That is not the Guruprasad instability index, which is computed from a table of dipeptide instability weights, and the threshold does not transfer.
The aliphatic index is shown instead. It is well defined, computed from a published formula, and measures the relative volume occupied by alanine, valine, isoleucine and leucine side chains, which correlates with thermostability. It is a real statistic reported as itself.
How each property is calculated
Six independent calculations over the parsed sequence, all using the same residue tables and the same pKa set as every other Volta tool that reports a charge or a weight.
MW = SUM(residue masses) + 18.0153
charge(pH)= SUM over basic groups of 1/(1+10^(pH-pKa))
- SUM over acidic groups of 1/(1+10^(pKa-pH))
pI = the pH at which charge(pH) = 0, by bisection
GRAVY = mean Kyte-Doolittle value per residue
epsilon280= 5500 x Trp + 1490 x Tyr + 125 x cystines
aliphatic = A% + 2.9 x V% + 3.9 x (I% + L%)- Parse and validate. Only the twenty standard single-letter codes are kept. Everything else is discarded so that no non-residue character can silently affect a count or a percentage.
- Sum residue masses. Average masses from the shared table, plus one water for the free termini.
- Apply Henderson-Hasselbalch per ionisable group. Both termini and every Asp, Glu, Cys, Tyr, His, Lys and Arg contribute a fractional charge determined by the difference between the pH and that group's pKa. The groups are treated as independent of each other.
- Bisect for the isoelectric point. The net charge is a monotonically falling function of pH, so a hundred rounds of bisection between pH 0 and 14 converge on the crossing point to well under a thousandth of a pH unit.
- Count chromophores for the extinction coefficient. The Pace method assigns 5,500 per tryptophan, 1,490 per tyrosine and 125 per disulfide bridge. Both the oxidised and the fully reduced figure are shown, because a sample in a reducing agent reads the lower one.
- Compute the aliphatic index. Mole percentages of alanine, valine, isoleucine and leucine weighted by the relative side chain volumes from Ikai's 1980 formula.
What this method cannot tell you
- •Charge and pI treat every ionisable group as independent. Neighbouring charges shift each other's pKa in real peptides, and the effect is largest in short, densely charged sequences.
- •Published pKa sets disagree with each other by up to a pH unit at the termini, which moves a calculated pI by a few tenths. Comparing pI values across tools that use different sets is not meaningful.
- •GRAVY is an average and hides the distribution. Use the hydrophobicity plotter for the profile.
- •Every figure describes the linear unmodified sequence. Terminal modifications, cyclisation and non-standard residues are all invisible to it.
Where the numbers come from
Peptide property calculator: frequently asked questions
The pH at which the peptide carries no net electrical charge, because its positive and negative groups exactly balance.
It is the pH at which solubility is lowest, because without net charge there is no electrostatic repulsion keeping molecules apart.
Because it tells you which pH to avoid. A peptide held at its pI is at the minimum of its solubility curve and the maximum of its aggregation risk.
Keep the working buffer at least one pH unit away from the pI. It also determines which ion exchange chemistry will bind the peptide, if you are purifying.
Each ionisable group contributes a fractional charge given by the Henderson-Hasselbalch relationship between the pH and that group's pKa, and the fractions are summed.
A group whose pKa equals the pH is exactly half ionised and contributes 0.5. Two pH units away it is 99 percent one way or the other, which is why net charge changes fastest near a pKa.
Because they use different published pKa sets, which disagree by up to a pH unit at the termini.
Every Volta tool uses one set so they agree with each other. A pI is an estimate with a spread of a few tenths, and comparing values across tools with different sets compares the sets rather than the peptides.
The grand average of hydropathy: the mean Kyte-Doolittle hydropathy value across every residue in the sequence.
- •Below about minus 1: strongly hydrophilic, reliably water-soluble
- •Around 0: mixed character, may need help dissolving
- •Above about plus 0.5: hydrophobic, usually needs a co-solvent
Yes, and this is the main weakness of an averaged score. A sequence with a strongly hydrophobic block and a strongly hydrophilic one averages near zero while behaving like neither.
The hydrophobicity plotter shows the value along the sequence rather than collapsed to a single number, which is the view that reveals this pattern.
The relative volume occupied by the side chains of alanine, valine, isoleucine and leucine, from Ikai's 1980 formula. Higher values correlate with greater thermostability.
A sequence of pure alanine scores exactly 100; one of pure valine scores 290; one with no aliphatic residues at all scores zero.
Because the figure that used to appear under that name was not the instability index. It was the percentage of D, P, S, T and G residues, presented with the 40-point threshold that belongs to the real statistic.
The Guruprasad instability index is computed from a table of dipeptide instability weights, which is a different calculation entirely. Rather than approximate it badly, this calculator reports the aliphatic index, which is well defined and computed exactly.
How strongly the peptide absorbs ultraviolet light at 280 nanometres, which determines whether you can measure its concentration in a spectrophotometer.
A peptide with no tryptophan, tyrosine or cysteine has a coefficient of zero and cannot be quantified at 280 nm at all. That is a common situation with short research peptides.
Because disulfide bridges contribute to absorbance and reducing agents break them. The oxidised value assumes every pair of cysteines is bridged; the reduced value assumes none are.
A sample in DTT or TCEP reads the lower figure. For peptides with no cysteine at all, the two are identical.
Partially. A strongly cationic peptide interacts with negatively charged membranes and surfaces, which is why cationic antimicrobial peptides both work the way they do and stick to labware.
It does not predict specific receptor binding, which depends on shape rather than on bulk charge.
Use it to rule buffers out. Any buffer whose working pH is within about one unit of the pI is the wrong choice for solubility.
The pH buffer calculator shows which systems cover which pH ranges, so a target well away from the pI can be matched to a buffer that actually buffers there.
Partly. Composition-based figures such as GRAVY and the aliphatic index are unaffected by cyclisation. Mass and charge are.
Head-to-tail cyclisation removes a water and eliminates both charged termini, which shifts the pI substantially in a short peptide where the termini are a large fraction of the total charge.
Only the twenty standard single-letter codes map to residue data. B, J, O, U, X and Z are ambiguity or rare-residue codes with no defined mass or pKa here, so they are discarded.
Discarding rather than substituting is deliberate: a guessed residue would change every number on the page without saying so.
Yes. The formulas are the same ones ProtParam applies to proteins, and they were developed on proteins.
They are in fact more reliable for larger sequences, because the independent-groups assumption behind the charge calculation holds better when no single group dominates.
Typically within a few tenths of a pH unit of a measured value for an unmodified peptide, and less reliable for short, densely charged sequences where neighbouring groups shift each other's pKa.
Treat it as a guide to which pH range to avoid rather than as a measurement.
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