title: Numerical and experimental demonstrations of the need for caution in the use of zonal gel chromatography for characterizing ligand interactions with small acceptors.
abstract: Quantitative expressions are presented for the evaluation of equilibrium constants for interactions of the type A + B in equilibrium C from experiments entailing the application of a small zone of acceptor-ligand mixture to a column of gel preequilibrated with ligand solution [J.P. Hummel and W.J. Dreyer (1962) Biochim. Biophys. Acta 63, 530-532]. Only in the event that identical elution volumes pertain to acceptor and complex does the steady-state binding constant (Kss) obtained by that method equal the thermodynamic equilibrium constant (K). Simulated elution profiles are then generated with parameters relevant to gel chromatography of the ATP-Mg2+ system on Sephadex G-10 in order to demonstrate the practical importance of the need for distinction between Kss and K in situations where acceptor and complex do not comigrate. A study of the interaction between soybean trypsin inhibitor and cytochrome c by gel chromatography on Sephadex G-75 is then used to illustrate the feasibility of combining information from Hummel-Dreyer experiments with the theoretical expressions to characterize systems under the more general conditions that the elution volumes of A and C differ. A finding of considerable theoretical interest in relation to the simulation of mass migration behavior is the demonstration that a truncation error is the source of zonal spreading in the theoretical-plate model of chromatography. This truncation error is shown to be the source of spreading generated whenever solution of an abbreviated (diffusion-free) continuity equation involves substituting first differences for first derivatives in the differential equation describing mass transport.
authors.affiliation: Department of Biochemistry/Biophysics/Genetics, University of Colorado Health Sciences Center, Denver.
authors.forename: J R
authors.initials: JR
authors.lastname: Cann
authors.forename: A G
authors.initials: AG
authors.lastname: Rao
authors.forename: D J
authors.initials: DJ
authors.lastname: Winzor
mesh_terms: D000255:Adenosine Triphosphate / Q000032:analysis; D002614:Chelating Agents / Q000032:analysis*; D002850:Chromatography, Gel / Q000379:methods*; D003574:Cytochrome c Group / Q000032:analysis; D007700:Kinetics; D008024:Ligands / Q000032:analysis*; D008274:Magnesium / Q000032:analysis; D008433:Mathematics; D008962:Models, Theoretical; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000032:analysis
chemical_list: D002614:Chelating Agents; D003574:Cytochrome c Group; D008024:Ligands; D000255:Adenosine Triphosphate; D014360:Trypsin Inhibitor, Kunitz Soybean; D008274:Magnesium