Mean Net Charge of Intrinsically Disordered Proteins: Experimental Determination of Protein Valence by Electrophoretic Mobility Measurements

  • Ana Cristina Sotomayor-Pérez
  • Johanna C. Karst
  • Daniel Ladant
  • Alexandre Chenal
Part of the Methods in Molecular Biology book series (MIMB, volume 896)


Under physiological conditions, intrinsically disordered proteins (IDPs) are unfolded, mainly because of their low hydrophobicity and the strong electrostatic repulsion between charged residues of the same sign within the protein. Softwares have been designed to facilitate the computation of the mean net charge of proteins (formally protein valence) from their amino acid sequences. Nevertheless, discrepancies between experimental and computed valence values for several proteins have been reported in the literature. Hence, experimental approaches are required to obtain accurate estimation of protein valence in solution. Moreover, ligand-induced disorder-to-order transition is involved in the folding of numerous IDPs. Some of the ligands are cations or anions, which, upon protein binding, decrease the mean net charge of the protein, favoring its folding via a charge reduction effect. An accurate determination of the mean net charge of protein in both its ligand-free intrinsically disordered state and in its folded, ligand-bound state allows one to estimate the number of ligands bound to the protein in the holo-state. Here, we describe an experimental protocol to determine the mean net charge of protein, from its electrophoretic mobility, its molecular mass and its hydrodynamic radius.

Key words

Mean net charge Protein valence Intrinsically disordered protein IDP Electrophoretic mobility Molecular mass Hydrodynamic radius Static light scattering Quasi-elastic light scattering Analytical ultracentrifugation 


\( {\mu_e} \)

Electrophoretic mobility, cm2.V−1.s−1 or μ−1.s−1 (SI: m2.V−1.s−1)

\( \zeta \)

Zeta potential, V


Valence or “mean net charge”


Electronic charge, 1.602 × 10−19 coulombs, A.s


Applied voltage, V, kg.m2.s−3.A−1


Current intensity, A

\( {\varepsilon_0} \)

Vacuum permittivity, 8.854 × 10−12C.V−1.m−1

\( {\varepsilon_r} \)

Relative static permittivity (dielectric constant) of the solvent, 78.54.

\( \varepsilon \)

Buffer permittivity, \( \varepsilon = {\varepsilon_r}{\varepsilon_0} \), C.V−1.m−1

\( {{f} \left/ {{{f_0}}} \right.} \)

Translational frictional ratio of the protein, including shape and hydration parameters

\( f \)

Frictional coefficient of the protein, g.s−1

\( {f_0} \)

Frictional coefficient of an anhydrous sphere of the mass of the protein, g.s−1


Hydrodynamic radius of the protein, cm


Hydrodynamic radius of the buffer, cm


Radius of an anhydrous sphere of the mass of the protein, cm


Hydrodynamic volume, cm3


Translational diffusion coefficient, cm2.s−1


Sedimentation coefficient obtained at the temperature of the experiment, Svedberg, 10−13s

\( \bar{\nu } \)

Partial specific volume, cm3.g−1

\( {\eta_s} \)

Viscosity of the solvent, Poise:−1.s−1

\( \rho \)

Density of the solvent,−3


Molecular mass, g.mol−1


Absolute temperature, K


Protein concentration, mol.L−1 (M)

\( \delta \)

Time-averaged apparent hydration, \( {g_{{{{\rm{H}}_{{2}}}{\rm{O}}}}} \times g_{\rm{protein}}^{{ - 1}} \)


Mean count rate


Kilo-count per second


Zeta quality factor


Fast field reversal


Slow field reversal


Malvern electrophoretic mobility cell


Malvern standard for electrophoretic mobility and conductivity


Quasi-elastic light scattering


Boltzmann’s constant, erg.K−1; (K B: 1.38065 × 10−16 erg.K−1 with erg: g.cm2.s−2 = 10−7 J; 1.38065 × 10−23 J.K−1)


Avogadro’s number, molecules.mol−1


Phase shift analysis light scattering


Debye length, Inverse screening length, m


Malvern electrophoretic mobility microcell



This work was supported by the Institut Pasteur (Grant PTR374), the Centre National de la Recherche Scientifique (CNRS UMR 3528), and the Agence Nationale de la Recherche, programme Jeunes Chercheurs (ANR, grant ANR-09-JCJC-0012).


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Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Ana Cristina Sotomayor-Pérez
    • 1
    • 2
  • Johanna C. Karst
    • 1
    • 2
  • Daniel Ladant
    • 1
    • 2
  • Alexandre Chenal
    • 1
    • 2
  1. 1.Unité de Biochimie des Interactions Macromoléculaires, CNRS UMR 3528, Institut PasteurParisFrance
  2. 2.Département de Biologie Structurale et Chimie, CNRS UMR 3528Institut Pasteur, Unité de Biochimie des Interactions MacromoléculairesParisFrance

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