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Abstract

Arrhenius plots of chloride and bromide transport yield two regions with different activation energies (E_a). Below 15 or 25°C (for Cl⁻ and Br⁻, respectively), E_a is about 32.5 kcal/mol; above these temperatures, about 22.5 kcal/mol (Brahm, J. (1977) J. Gen. Physiol. 70, 283–306). For the temperature dependence of SO₄²⁻ transport up to 37°C, no such break could be observed. We were able to show that the temperature coefficient for the rate of SO₄²⁻ transport is higher than that for the rate of denaturation of the band 3 protein (as measured by NMR) or the destruction of the permeability barrier in the red cell membrane. It was possible, therefore, to extend the range of flux measurements up to 60°C and to show that, even for the slowly permeating SO₄²⁻ in the Arrhenius plot, there appears a break, which is located somewhere between 30 and 37°C and where E_a changes from 32.5 to 24.1 kcal/mol. At the break, the turnover number is approx. 6.9 ions/band 3 per s. Using ³⁵Cl⁻-NMR (Falke, Pace and Chan (1984) J. Biol. Chem. 259, 6472–6480), we also determined the temperature dependence of Cl⁻-binding. We found no significant change over the entire range from 0 to 57°C, regardless of whether the measurements were performed in the absence or presence of competing SO₄²⁻. We conclude that the enthalpy changes associated with Cl⁻-or SO4²⁻-binding are negligible as compared to the E_a values observed. It was possible, therefore, to calculate the thermodynamic parameters defined by transition-state theory for the transition of the anion-loaded transport protein to the activated state for Cl⁻, Br⁻ and SO₄²⁻ below and above the temperatures at which the breaks in the Arrhenius plots are seen. We found in both regions a high positive activation entropy, resulting in a low free enthalpy of activation. Thus the internal energy required for carrying the complex between anion and transport protein over the rate-limiting energy barrier is largely compensated for by an increase of randomness in the protein and/or its aqueous environment.