| This work continues the development of a new method for measurement of ligand and macromolecule interaction called pulsed ultrafiltration.;We have applied the pulsed ultrafiltration method to a system with such a well defined binding site--RNase A and its inhibitor 2;The ease with which the pulsed ultrafiltration method can be applied to binding measurements suggested that it could be useful to thermodynamic measurements. The entropy and enthalpy changes of RNase A upon the binding of 3;We have also developed a mathematical model applicable to competitive binding studies using the pulsed ultrafiltration method. Our study of warfarin/phenylbutazone competitive binding to HSA demonstrates that the pulsed ultrafiltration method gives an accurate analysis of competitive binding as well.;In the present work, a mathematical treatment of elution profiles developed from basic kinetic principles is presented. The solution of a set of differential equations describes the ligand elution for ligand/macromolecule binding with 1:1 stoichiometry. Experimentally, measurement of binding constant for a Trp/BSA system obtained using this differential method gives the same result as the earlier developed area integration method and consists with literature reports.;In conclusion, we have developed a new method for measuring ligand/macromolecule interactions. It conserves ligand and macromolecule while yielding a continuous set of data points across on entire binding isotherm. This method can be adapted to temperature dependency studies to obtain entropy and enthalpy changes upon the binding, as well as the competitive binding studies. It is a rapid, convenient, reliable, and accurate method for measuring binding parameters, and that its capabilities may actually exceed those of the more classical approaches. |
