Determination of effective protein charge by capillary electrophoresis: Effects of charge regulation in the analysis of charge ladders

M. K. Menon, Andrew Zydney

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

Protein charge ladders are an effective tool for measuring protein charge and studying electrostatic interactions. However, previous analyses have neglected the effects of charge regulation, the alteration in the extent of amino acid ionization associated with differences between the pH at the protein surface and in the bulk solution. Experimental data were obtained with charge ladders constructed from bovine carbonic anhydrase. The protein charge for each element in the ladder was calculated from the protein electrophoretic mobility as measured by capillary electrophoresis using the hindrance factor for a hard sphere with equivalent hydrodynamic radius. The protein charge was also evaluated theoretically from the amino acid sequence by assuming a Boltzmann distribution in the hydrogen ion concentration. The calculations were in excellent agreement with the data, demonstrating the importance of charge regulation on the net protein charge. These results have important implications for the use of charge ladders to evaluate effective protein charge in solution.

Original languageEnglish (US)
Pages (from-to)5714-5717
Number of pages4
JournalAnalytical Chemistry
Volume72
Issue number22
DOIs
StatePublished - Nov 15 2000

Fingerprint

Capillary electrophoresis
Ladders
Proteins
pH
Amino Acids
Electrophoretic mobility
Carbonic Anhydrases
Coulomb interactions
Ionization
Membrane Proteins
Hydrodynamics

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

Cite this

@article{c0dbea181e0a4af19b892d3363d3d100,
title = "Determination of effective protein charge by capillary electrophoresis: Effects of charge regulation in the analysis of charge ladders",
abstract = "Protein charge ladders are an effective tool for measuring protein charge and studying electrostatic interactions. However, previous analyses have neglected the effects of charge regulation, the alteration in the extent of amino acid ionization associated with differences between the pH at the protein surface and in the bulk solution. Experimental data were obtained with charge ladders constructed from bovine carbonic anhydrase. The protein charge for each element in the ladder was calculated from the protein electrophoretic mobility as measured by capillary electrophoresis using the hindrance factor for a hard sphere with equivalent hydrodynamic radius. The protein charge was also evaluated theoretically from the amino acid sequence by assuming a Boltzmann distribution in the hydrogen ion concentration. The calculations were in excellent agreement with the data, demonstrating the importance of charge regulation on the net protein charge. These results have important implications for the use of charge ladders to evaluate effective protein charge in solution.",
author = "Menon, {M. K.} and Andrew Zydney",
year = "2000",
month = "11",
day = "15",
doi = "10.1021/ac000752b",
language = "English (US)",
volume = "72",
pages = "5714--5717",
journal = "Analytical Chemistry",
issn = "0003-2700",
publisher = "American Chemical Society",
number = "22",

}

Determination of effective protein charge by capillary electrophoresis : Effects of charge regulation in the analysis of charge ladders. / Menon, M. K.; Zydney, Andrew.

In: Analytical Chemistry, Vol. 72, No. 22, 15.11.2000, p. 5714-5717.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Determination of effective protein charge by capillary electrophoresis

T2 - Effects of charge regulation in the analysis of charge ladders

AU - Menon, M. K.

AU - Zydney, Andrew

PY - 2000/11/15

Y1 - 2000/11/15

N2 - Protein charge ladders are an effective tool for measuring protein charge and studying electrostatic interactions. However, previous analyses have neglected the effects of charge regulation, the alteration in the extent of amino acid ionization associated with differences between the pH at the protein surface and in the bulk solution. Experimental data were obtained with charge ladders constructed from bovine carbonic anhydrase. The protein charge for each element in the ladder was calculated from the protein electrophoretic mobility as measured by capillary electrophoresis using the hindrance factor for a hard sphere with equivalent hydrodynamic radius. The protein charge was also evaluated theoretically from the amino acid sequence by assuming a Boltzmann distribution in the hydrogen ion concentration. The calculations were in excellent agreement with the data, demonstrating the importance of charge regulation on the net protein charge. These results have important implications for the use of charge ladders to evaluate effective protein charge in solution.

AB - Protein charge ladders are an effective tool for measuring protein charge and studying electrostatic interactions. However, previous analyses have neglected the effects of charge regulation, the alteration in the extent of amino acid ionization associated with differences between the pH at the protein surface and in the bulk solution. Experimental data were obtained with charge ladders constructed from bovine carbonic anhydrase. The protein charge for each element in the ladder was calculated from the protein electrophoretic mobility as measured by capillary electrophoresis using the hindrance factor for a hard sphere with equivalent hydrodynamic radius. The protein charge was also evaluated theoretically from the amino acid sequence by assuming a Boltzmann distribution in the hydrogen ion concentration. The calculations were in excellent agreement with the data, demonstrating the importance of charge regulation on the net protein charge. These results have important implications for the use of charge ladders to evaluate effective protein charge in solution.

UR - http://www.scopus.com/inward/record.url?scp=0034669239&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034669239&partnerID=8YFLogxK

U2 - 10.1021/ac000752b

DO - 10.1021/ac000752b

M3 - Article

C2 - 11101252

AN - SCOPUS:0034669239

VL - 72

SP - 5714

EP - 5717

JO - Analytical Chemistry

JF - Analytical Chemistry

SN - 0003-2700

IS - 22

ER -