Temperature dependence of anion transport inhibitor binding to human red cell membranes

Richard G Posner, James A. Dix

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

The binding characteristics of the inhibitor of anion transport in human red cells, 4,4′- dibenzamido-2,2′-disulfonic stilbene (DBDS), to the anion transport protein of red cell ghost membranes in buffer containing 150 mM NaCl have been measured over the temperature range 0-30°C by equilibrium and stopped-flow fluorescence methods. The equilibrium dissociation constant, Keq, increased with temperature. No evidence of a 'break' in the ln(Keq) vs. 1/T plot was found. The standard dissociation enthalpy and entropy changes calculated from the temperature dependence are 9.1 ± 0.9 kcal/mol and 3.2 ± 0.3 e.u., respectively. Stopped-flow kinetic studies resolve the overall binding into two steps: a bimolecular association of DBDS with the anion transport protein, followed by a unimolecular rearrangement of the DBDS-protein complex. The rate constants for the individual steps in the binding mechanism can be determined from an analysis of the concentration dependence of the binding time course. Arrhenius plots of the rate constants showed no evidence of a break. Activation energies for the individual steps in the binding mechanism are 11.6 ± 0.9 kcal/mol (bimolecular, forward step), 17 ± 2 kcal/mol (bimolecular, reverse step), 6.4 ± 2.3 kcal/mol (unimolecular, forward step), and 10.6 ± 1.9 kcal/mol (unimolecular, reverse step). Our results indicate that there is an appreciable enthalpic energy barrier for the bimolecular association of DBDS with the transport protein, and appreciable enthalpic and entropic barriers for the unimolecular rearrangement of the DBDS-protein complex.

Original languageEnglish (US)
Pages (from-to)139-145
Number of pages7
JournalBiophysical Chemistry
Volume23
Issue number1-2
DOIs
StatePublished - 1985
Externally publishedYes

Fingerprint

Stilbenes
Cell membranes
inhibitors
Anions
stilbene
Cell Membrane
Anion Transport Proteins
anions
temperature dependence
Temperature
proteins
Rate constants
Association reactions
Arrhenius plots
Energy barriers
Erythrocyte Membrane
plots
Entropy
dissociation
Enthalpy

Keywords

  • 4,4′- Dibenzamido - 2,2′- disulfonic stilbene
  • Anion transport
  • Band 3
  • Red cell membrane
  • Thermodynamics

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Physical and Theoretical Chemistry

Cite this

Temperature dependence of anion transport inhibitor binding to human red cell membranes. / Posner, Richard G; Dix, James A.

In: Biophysical Chemistry, Vol. 23, No. 1-2, 1985, p. 139-145.

Research output: Contribution to journalArticle

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AB - The binding characteristics of the inhibitor of anion transport in human red cells, 4,4′- dibenzamido-2,2′-disulfonic stilbene (DBDS), to the anion transport protein of red cell ghost membranes in buffer containing 150 mM NaCl have been measured over the temperature range 0-30°C by equilibrium and stopped-flow fluorescence methods. The equilibrium dissociation constant, Keq, increased with temperature. No evidence of a 'break' in the ln(Keq) vs. 1/T plot was found. The standard dissociation enthalpy and entropy changes calculated from the temperature dependence are 9.1 ± 0.9 kcal/mol and 3.2 ± 0.3 e.u., respectively. Stopped-flow kinetic studies resolve the overall binding into two steps: a bimolecular association of DBDS with the anion transport protein, followed by a unimolecular rearrangement of the DBDS-protein complex. The rate constants for the individual steps in the binding mechanism can be determined from an analysis of the concentration dependence of the binding time course. Arrhenius plots of the rate constants showed no evidence of a break. Activation energies for the individual steps in the binding mechanism are 11.6 ± 0.9 kcal/mol (bimolecular, forward step), 17 ± 2 kcal/mol (bimolecular, reverse step), 6.4 ± 2.3 kcal/mol (unimolecular, forward step), and 10.6 ± 1.9 kcal/mol (unimolecular, reverse step). Our results indicate that there is an appreciable enthalpic energy barrier for the bimolecular association of DBDS with the transport protein, and appreciable enthalpic and entropic barriers for the unimolecular rearrangement of the DBDS-protein complex.

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