Isoelectric point calculation of native proteins

The isoelectric point (pI) of native (not denaturated) proteins is important in several separation techniques. The pI is defined as the pH value at which the positive and negative charges on the protein are balanced and the net charge is zero. To determine the pH at which the net charge is zero, the charge on the protein is calculated at multiple pH values using Henderson-Hasselbalch equation, as described by Sillero and Ribeiro (1989).

Charge of type N (negative) groups:

Basic charges

Charge of type P (positive) groups:

Acidic charges

Total charge:

Total charges

The usage of appropiate pK values is of crucial importance to caluclate correct pI. For denatured proteins, Bjellqvist et al. (1993) published an accurate methode to predict the focusing position of polypeptide in immmobilized pH gradients under denaturing conditions (8 M urea). On the other hand, amino acid side chain pK values of native proteins where measured using NMR. Grimsley et al. (2008) summarized 541 pK values of 78 folded proteins measured with NMR which were taken to calculate average pK values for each type of amino acid side chain. An algorithm to determine the isoelectric point of native protein using these average pK values is implemented in Prot pi’s Protein Tool. Choose “native” option in the “Advanced” tab to take advantage of this algorithm (figure 1).

Figure 1: How to use the algorithm to calculate isoelectric points of native proteins.
Figure 1: How to use the algorithm to calculate isoelectric points of native proteins.

The calculated and the measured isoelectric point of five proteins with different properties were compared. Homo-and heteromultimeric, basic and acidic as well as small and very large proteins were taken to validate the calculated pI (table 1).

Table 1: Calculated pI compared with measured pI out of literature of native and denaturated proteins. Measured pI were taken from Ui (2006), except: *) Przybylski (2015) and **) Stone-Wolff (1984)
ProteinUniprot #pI, measured, nativepI, measured, denaturatedpI,
calculated, native
calculated, denatured
5.725.765.5355.438Disulfide linked heterodimer
Ribonuclease>sp|P61823|27-1509.339.589.7439.19Basic pI
Thyroglobulin>tr|D1KKB3|20-26924.55.35.5345.433Very large (582.5 kDa)
Serum albumin>sp|P02768|25-6095.85.95.8615.724Acidic pI

The calculated pI values showed reasonably good agreement with experimental ones for all of the six proteins under native and denaturing conditions over a wide range of physico-chemical properties (figure 2).

Figure 2: Correlation of experimental and calculated isoelectric point (pI) of native (blue) and denaturated (green) proteins.
Figure 2: Correlation of experimental and calculated isoelectric point (pI) of native (blue) and denaturated (green) proteins.


Prot pi provides an accurate tool to calculate isoelectric points of denatured and native proteins that takes charge contributions of multiple subunits, disulfide bonds and posttranslational modifications into account.

About Roland Josuran

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13. March 2021 20:28

I Don’t know if my computer is glitching, but your profile picture is on top of “Author’s name”.

Prot pi
15. March 2021 17:38
Reply to  colin

Thanks for reporting this! The issue was caused by an outdated setting, which is now fixed.

19. December 2021 6:10

Hello, how good night, in which menu can I know the isoelectric point of my denatured protein and which parameter to take from my isoelectric point the first without placing the native option or should I select native?

Prot pi
20. December 2021 9:54
Reply to  Reguinaldo

Dear Reguinaldo
For denatured proteins it is best to use the “ProMoST” option, which is the standard.

Lun Jiang
Lun Jiang
8. May 2023 10:02

Hello, the isoelectric point of my protein was different in prediction with ProMost, Expasy and Native. Which one should I used.

Prot pi
8. June 2023 15:42
Reply to  Lun Jiang

If you are not sure which algorithm to use, you are certainly well advised to use ProMoST. It usually provides the most accurate results.