Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor

Biochemistry. 2005 Apr 5;44(13):5258-66. doi: 10.1021/bi048135v.


ThermoFluor (a miniaturized high-throughput protein stability assay) was used to analyze the linkage between protein thermal stability and ligand binding. Equilibrium binding ligands increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. Binding constants (K(b)) were measured by examining the systematic effect of ligand concentration on protein stability. The precise ligand effects depend on the thermodynamics of protein stability: in particular, the unfolding enthalpy. An extension of current theoretical treatments was developed for tight binding inhibitors, where ligand effect on T(m) can also reveal binding stoichiometry. A thermodynamic analysis of carbonic anhydrase by differential scanning calorimetry (DSC) enabled a dissection of the Gibbs free energy of stability into enthalpic and entropic components. Under certain conditions, thermal stability increased by over 30 degrees C; the heat capacity of protein unfolding was estimated from the dependence of calorimetric enthalpy on T(m). The binding affinity of six sulfonamide inhibitors to two isozymes (human type 1 and bovine type 2) was analyzed by both ThermoFluor and isothermal titration calorimetry (ITC), resulting in a good correlation in the rank ordering of ligand affinity. This combined investigation by ThermoFluor, ITC, and DSC provides a detailed picture of the linkage between ligand binding and protein stability. The systematic effect of ligands on stability is shown to be a general tool to measure affinity.

MeSH terms

  • Animals
  • Calorimetry, Differential Scanning
  • Carbonic Anhydrase I / chemistry
  • Carbonic Anhydrase I / metabolism
  • Carbonic Anhydrase II / chemistry
  • Carbonic Anhydrase II / metabolism
  • Carbonic Anhydrase Inhibitors / chemistry
  • Carbonic Anhydrases / chemistry*
  • Carbonic Anhydrases / metabolism*
  • Cattle
  • Enzyme Stability
  • Humans
  • In Vitro Techniques
  • Kinetics
  • Protein Denaturation
  • Spectrometry, Fluorescence
  • Thermodynamics


  • Carbonic Anhydrase Inhibitors
  • Carbonic Anhydrase I
  • Carbonic Anhydrase II
  • Carbonic Anhydrases