### 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 = C_{P}Z(MW)^{-α}, C_{P} = 6.3 cm^{2} min^{-1} kV^{-1} charge^{-1} kD^{0.48}, α = 0.48; for μ = C_{r}Z[r(1 + kr)]^{-1}, C_{r} - 55 cm^{2} 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 language | English (US) |
---|---|

Pages (from-to) | 575-580 |

Number of pages | 6 |

Journal | Analytical Chemistry |

Volume | 69 |

Issue number | 4 |

State | Published - 1997 |

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### ASJC Scopus subject areas

- Analytical Chemistry

### Cite this

*Analytical Chemistry*,

*69*(4), 575-580.

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

Research output: Contribution to journal › Article

*Analytical Chemistry*, vol. 69, no. 4, pp. 575-580.

}

TY - JOUR

T1 - Using Protein Charge Ladders to Estimate the Effective Charges and Molecular Weights of Proteins in Solution

AU - Gao, Jinming

AU - Whitesides, George M.

PY - 1997

Y1 - 1997

N2 - 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)-α, 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.

AB - 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)-α, 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.

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M3 - Article

VL - 69

SP - 575

EP - 580

JO - Analytical Chemistry

JF - Analytical Chemistry

SN - 0003-2700

IS - 4

ER -