Using Protein Charge Ladders to Estimate the Effective Charges and Molecular Weights of Proteins in Solution

Jinming Gao, George M. Whitesides

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Abstract

This paper describes the use of capillary electrophoresis (CE) and protein charge ladders to estimate values of effective charge (Z) and molecular weight of proteins under nondenaturing conditions. A panel of 14 proteins with a range of charges and shapes was modified by accetylation with acetic anhydride to yield protein charge ladders. A protein charge ladder is a family of derivatives of a protein that differ in integral units charge, but minimally in hydrodynamic drag; this mixture of proteins appears in electrophoresis as a set of peaks with regular spacing. Analysis of the electrophoretic mobilities of the members of these charge ladders yields values of Z and electrophoretic coefficients: for a description of mobility based on the equation μ = CPZ(MW), CP = 6.3 cm2 min-1 kV-1 charge-1 kD0.48, α = 0.48; for μ = CrZ[r(1 + kr)]-1, Cr - 55 cm2 min-1 kV-1 charge-1 Å(r is the spherical radius of the protein and k is a function of ionic strength). The primary usefulness of charge ladders is to measuring the effective charge, Z, of proteins in solution; this information is difficult to obtain by any other procedure. A secondary value of the method is to estimate values of molecular weight. Although less general and convenient than SDS-PAGE, this method allows estimates of molecular weight of nondenatured proteins and is thus applicable to oligomers, noncovalent aggregates, proteins with multiple, non-cross-linked chains, and other systems to which SDS-PAGE is not applicable. The values of molecular weight calculated using the electrophoretic mobilities of proteins in solution and the above constants agreed with literature values to within 20% (with an ambiguous result for ovalbumin). A combination of this technique and SDS-PAGE will be useful in estimating the number of subunits or stage of aggregation of proteins in solution.

Original languageEnglish (US)
Pages (from-to)575-580
Number of pages6
JournalAnalytical Chemistry
Volume69
Issue number4
StatePublished - 1997

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Ladders
Molecular weight
Proteins
Electrophoretic mobility
Capillary electrophoresis
Ovalbumin
Ionic strength
Electrophoresis
Oligomers
Drag
Agglomeration
Hydrodynamics
Derivatives

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Using Protein Charge Ladders to Estimate the Effective Charges and Molecular Weights of Proteins in Solution. / Gao, Jinming; Whitesides, George M.

In: Analytical Chemistry, Vol. 69, No. 4, 1997, p. 575-580.

Research output: Contribution to journalArticle

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