Notes

This page describes some technical details of our Peptide Calculator.

Calculating the molecular weight

The molecular weight M of a peptide may be estimated by calculating

The equation for the molecular weight of a peptide.

where N_i are the number, and M_i the average residue molecular weights, of the amino acids. M_N + M_C are added to the total in order to account for the termini: H at the N-terminus and OH at the C-terminus. Of course, if the termini are modified, these additions are replaced by those of the modifiers.
The resulting molecular weight depends on what values the algorithm uses. Some of the values Innovagen's Peptide Calculator uses are:

A, Ala	71.07793	C, Cys	103.1454	D, Asp	115.0873	E, Glu	129.1139
F, Phe	147.1734	G, Gly	57.05138	H, His	137.1394	I, Ile	113.1576
K, Lys	128.1724	L, Leu	113.1576	M, Met	131.1985	N, Asn	114.1028
P, Pro	97.11508	Q, Gln	128.1293	R, Arg	156.1861	S, Ser	87.07733
T, Thr	101.1039	V, Val	99.13103	W, Trp	186.2095	Y, Tyr	163.1728

H	1.00797	OH	17.00738			phos-Ser	167.0573
Acetyl	43.04453	Amide	16.0228			phos-Thr	181.0838
Biotin	227.3056					phos-Tyr	243.1528

Calculating the extinction coefficient

The molar extinction coefficient e at 280 nm of a peptide can be estimated by calculating

The equation for the extinction coefficient of a peptide.

where n_W, n_Y, and n_C are the number of Tryptophans (W), Tyrosines (Y) and Cystines (i.e. disulphide bonds, but here denoted C) in the sequence. The molar extinction coefficients used in Innovagen's Peptide Calculator are:

e_W = 5690 M^-1cm^-1	e_Y = 1280 M^-1cm^-1	e_C = 120 M^-1cm^-1

Gill and von Hippel

Note: Your peptide may contain non-natural amino acids, modifications and labels. None of these are considered, although they may very well add to the extinction coefficient.

Calculating the net charge

The net charge Z of a peptide at a certain pH can be estimated by calculating

The equation for the net charge of a peptide.

where N_i are the number, and pKa_i the pKa values, of the N-terminus and the side chains of Arginine, Lysine, and Histidine. The j-index pertain to the C-terminus and the Aspartic Acid, Glutamic Acid, Cysteine, Tyrosine amino acids.

Innovagen's Peptide Property Calculator calculates the net charge for all pH values of 0.1 to 14 in increments of 0.1, and plots these producing a titration curve.

This algorithm has its limitations, some of which are:

The residues are assumed to be independent of each other.
Only free terminii and the 20 naturally occuring amino acids and their D-forms are considered, all others are ignored.
The resulting net charge depends on what pKa values the algorithm uses. Innovagen's Peptide Property Calculator uses the values taken from the CRC Handbook of Chemistry and Physics, 87th edition.

Calculating the isoelectric point, pI

The isoelectric point, pI, is the pH at which the net charge of the peptide is zero. Innovagen's Peptide Calculator first calculates the net charge for pH 7.0. If the charge is > 0 the next pH to check is 7 + 3.5, if the charge is < 0 then pH 7 - 3.5 is checked. This is repeated, using increments/decrements half the size of the previous, until the charge found equals 0, or is sufficiently close to 0.

Hydrophathy

Innovagen's Peptide Calculator plots the hydrophathy for each standard amino acid in the peptide sequence in a colour coded bar graph. Hydrophilic residues are shown as bars rising upwards, while hydrophobic have downwards bars. The results depend on what index is used. Innovagen's Peptide Calculator uses the following values:

Solubility

Please note that the result presented in this Peptide Calculator is only a very rough estimation of water solubility. Determining the solubility of a peptide must be done by experiment. For example, if we enter the sequence for the beta amyloid 1-40 peptide, the result will be "good water solubility". In practice, this peptide will dissolve properly at an elevated pH.

Guide to solubilising your peptide

If your application allows for it, then using an organic solvent like DMSO is a convenient way of dissolving your peptide. Once the peptide is in solution, it can be diluted further with pure water.

Most peptides will dissolve in pure water. Any buffer components that are required can then typically be added afterwards.

When a peptide won't dissolve in pure water, and organic solvents like DMSO are not an option, then there are several things to try.

When trying the latter option, shifting pH, the idea is to move away from the iso-electric point. If you have an acidic peptide, i.e. where the iso-electric point is <7 you should raise the pH, and conversely, when the pI is >7 the pH should be lowered. Taking beta amyloid 1-40 as an example again, the pI is 5.2 and as it happens this peptide dissolves at pH 9 but not at pH 7.

A, Ala	-0.5	C, Cys	-1	D, Asp	3	E, Glu	3
F, Phe	-2.5	G, Gly	0	H, His	-0.5	I, Ile	-1.8
K, Lys	3	L, Leu	-1.8	M, Met	-1.3	N, Asn	0.2
P, Pro	0	Q, Gln	0.2	R, Arg	3	S, Ser	0.3
T, Thr	-0.4	V, Val	-1.5	W, Trp	-3.4	Y, Tyr	-2.3

Hopp & Woods