Capillary-zone electrophoresis of ds-DNA in isoelectric buffers

Effect of adding of competing, nonamphoteric ions

Cecilia Gelfi, Daniela Mauri, Massimiliano Perduca, Nancy C. Stellwagen, Pier Giorgio Righetti

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

When separating ds-DNA in isoelectric His buffer (pH=pI=7.6), in the 50-250 mM concentration range, some unique phenomena were observed: improved resolution for smaller DNA fragments, up to ca. 150 bp, and a rapid deterioration of resolution above this critical length (which corresponds to the persistence length). Such phenomenon depended also on voltage and concentration of sieving liquid polymer. Direct binding of His to the DNA helix was hypothesized, with resultant stiffening and an increment of diameter of the DNA fragments, thus inducing an early onset of reptation at the applied voltage in the 100-300 V/cm range. In order to prove this hypothesis, 'competing ions' (notably NaCl and KBr) were added to the His background electrolyte: a partial reversal of the His effect was already apparent at low concentrations of such ions (10 mM) and was complete at higher concentrations (30 and 50 mM). By molecular modeling, it was found that His could be docking on the negatively charged oxygen (bound to the phosphate) by offering both charged (primary and tertiary amino) groups to simultaneous binding, thus forming a salt and neutralizing the negative charge borne by the oxygen. The following characteristic bond distances were found: 0.34 nm between the N (imidazolic) and O; 0.32 nm between the primary N and O; 0.36 nm between the two nitrogens engaged in salt formation with the oxygen. In addition, for complexation to occur, the distance between the noncharged nitrogen in the imidazole ring and the nearest phosphate oxygen (engaged in the phosphodiester bridge) should be 0.44 nm. Under these conditions, the two rings present (a six-membered, ideal one, salt-linked with the oxygen and rather highly elongated, and the imidazole) will not be precisely coplanar, since the primary and tertiary nitrogens will be one slightly above and one slightly below the plane of the drawing. Upon extensive binding, occupying every available phosphate site, π-π interactions could occur among the stacks of bound His residues, thus further stabilizing the complex.

Original languageEnglish
Pages (from-to)1704-1710
Number of pages7
JournalElectrophoresis
Volume19
Issue number10
Publication statusPublished - Jul 1998

Fingerprint

Capillary Electrophoresis
Electrophoresis
Buffers
Ions
Oxygen
DNA
Nitrogen
Salts
Phosphates
Molecular modeling
Electric potential
Complexation
Electrolytes
Deterioration
Polymers
Liquids
imidazole

Keywords

  • Capillary electrophoresis
  • Double-stranded DNA
  • Isoelectric buffers

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Gelfi, C., Mauri, D., Perduca, M., Stellwagen, N. C., & Righetti, P. G. (1998). Capillary-zone electrophoresis of ds-DNA in isoelectric buffers: Effect of adding of competing, nonamphoteric ions. Electrophoresis, 19(10), 1704-1710.

Capillary-zone electrophoresis of ds-DNA in isoelectric buffers : Effect of adding of competing, nonamphoteric ions. / Gelfi, Cecilia; Mauri, Daniela; Perduca, Massimiliano; Stellwagen, Nancy C.; Righetti, Pier Giorgio.

In: Electrophoresis, Vol. 19, No. 10, 07.1998, p. 1704-1710.

Research output: Contribution to journalArticle

Gelfi, C, Mauri, D, Perduca, M, Stellwagen, NC & Righetti, PG 1998, 'Capillary-zone electrophoresis of ds-DNA in isoelectric buffers: Effect of adding of competing, nonamphoteric ions', Electrophoresis, vol. 19, no. 10, pp. 1704-1710.
Gelfi, Cecilia ; Mauri, Daniela ; Perduca, Massimiliano ; Stellwagen, Nancy C. ; Righetti, Pier Giorgio. / Capillary-zone electrophoresis of ds-DNA in isoelectric buffers : Effect of adding of competing, nonamphoteric ions. In: Electrophoresis. 1998 ; Vol. 19, No. 10. pp. 1704-1710.
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abstract = "When separating ds-DNA in isoelectric His buffer (pH=pI=7.6), in the 50-250 mM concentration range, some unique phenomena were observed: improved resolution for smaller DNA fragments, up to ca. 150 bp, and a rapid deterioration of resolution above this critical length (which corresponds to the persistence length). Such phenomenon depended also on voltage and concentration of sieving liquid polymer. Direct binding of His to the DNA helix was hypothesized, with resultant stiffening and an increment of diameter of the DNA fragments, thus inducing an early onset of reptation at the applied voltage in the 100-300 V/cm range. In order to prove this hypothesis, 'competing ions' (notably NaCl and KBr) were added to the His background electrolyte: a partial reversal of the His effect was already apparent at low concentrations of such ions (10 mM) and was complete at higher concentrations (30 and 50 mM). By molecular modeling, it was found that His could be docking on the negatively charged oxygen (bound to the phosphate) by offering both charged (primary and tertiary amino) groups to simultaneous binding, thus forming a salt and neutralizing the negative charge borne by the oxygen. The following characteristic bond distances were found: 0.34 nm between the N (imidazolic) and O; 0.32 nm between the primary N and O; 0.36 nm between the two nitrogens engaged in salt formation with the oxygen. In addition, for complexation to occur, the distance between the noncharged nitrogen in the imidazole ring and the nearest phosphate oxygen (engaged in the phosphodiester bridge) should be 0.44 nm. Under these conditions, the two rings present (a six-membered, ideal one, salt-linked with the oxygen and rather highly elongated, and the imidazole) will not be precisely coplanar, since the primary and tertiary nitrogens will be one slightly above and one slightly below the plane of the drawing. Upon extensive binding, occupying every available phosphate site, π-π interactions could occur among the stacks of bound His residues, thus further stabilizing the complex.",
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