We propose to study the kinetics of a number of non-covalent complexes of small molecules with blood proteins. By a new method we expect to be able to study reactions for which existing techniques are not adequate. Ligands of interest include metabolites, hormones, antibiotics, and therapeutic as well as toxic drugs. The new technique consists in using rapid measurements of molecular dipole moment as a means of detecting the progress of such complexing reactions. It will be used in the first instance to study systems in which the binding reaction does not result in significant changes in optical parameters. We have found in preliminary studies that, for example, the complexing of one mole of fatty acid with human serum albumin results in a 30% change in the dielectric increment, while no optical parameters change. The complexing reaction is initiated in a rapid-flow apparatus, and the reacting mixture is injected between two concentric cylindrical electrodes, instead of into a conventional optical observation chamber. A measurement cell volume of 16 microliters has been found satisfactory. In the cell, the dielectric increment is measured by a pulse technique at short intervals to trace the rate of the reaction. For theoretical reasons, the method is expected to be an equally sensitive detector of short lived intermediate binding states.
NIH ExPorter
NIH ExPorter