Isothermal titration calorimetric procedure to determine protein-metal ion binding parameters in the presence of excess metal ion or chelator.
Determination of binding parameters for metal ion binding to proteins usually requires preceding steps to remove protein-bound metal ions. Removal of bound metal ions from protein is often associated with decreased stability and inactivation. We present two simple isothermal titration calorimetric procedures that eliminate separate metal ion removal steps and directly monitor the exchange of metal ions between buffer, protein, and chelator. The concept is to add either excess chelator or metal ion to the protein under investigation and subsequently titrate with metal ion or chelator, respectively. It is thereby possible in the same experimental trial to obtain both chelator-metal ion and protein-metal ion binding parameters due to the different thermodynamic "fingerprints" of chelator and protein. The binding models and regression routines necessary to analyze the corresponding binding isotherms have been constructed. Verifications of the models have been done by titrations of mixtures of calcium chelators (BAPTA, HEDTA, and EGTA) and calcium ions and they were both able to account satisfactorily for the observed binding isotherms. Therefore, it was possible to determine stoichiometric and thermodynamic binding parameters. In addition, the concept has been tested on a recombinant alpha-amylase from Bacillus halmapalus where it proved to be a consistent procedure to obtain calcium binding parameters.
Nielsen
Anders D
AD
Department of Life Sciences and Chemistry, Roskilde University, P. O. Box 260, DK-4000 Roskilde, Denmark.
Fuglsang
Claus C
CC
Westh
Peter
P
D001407:Bacillus / Q000235:genetics; D001665:Binding Sites / Q000187:drug effects; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D002151:Calorimetry / Q000379:methods*; D002614:Chelating Agents / Q000378:metabolism* / Q000494:pharmacology; D004492:Edetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D002614:Chelating Agents; D008024:Ligands; D011994:Recombinant Proteins; D004533:Egtazic Acid; D004492:Edetic Acid; D000516:alpha-Amylases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; C026060:N-(hydroxyethyl)ethylenediaminetriacetic acid; D002118:Calcium