Volume 4, Number 3, p. 145–156
Impact of experimental and instrumental conditions on the measured results of kinetic and equilibrium constant determinations involving biopolymers
Impact of experimental and instrumental conditions on the measured results of kinetic and equilibrium constant determinations involving biopolymers
C.J. van Oss,1,2,3 R.F. Giese2 and A. Docoslis4
1 Departments of Microbiology and Immunology, 2Chemical Engineering and Geology and 3Chemical Engineering, State University of New York at Buffalo, Buffalo, New York, USA;
4Department of Chemical Engineering, Queen’s University,Kingston, Ontario, Canada.
This paper describes how to avoid inherent errors in an experimental approach to the measurement of kinetic association rate constants (ka ) involving dissolved ligand molecules (such as antibodies or their paratopes, or other (bio)polymers, and fixed receptor sites such as antigens or their epitopes, or other attractive sites). These include measurements employing low concentrations of dissolved ligand molecules or other (bio)polymers and expressing the measured ka values only after extrapolation of the concentration of these dissolved ligands tends to zero. It is found that ka values also need to be measured within the shortest possible time-span after inception of the interaction (typically within less than one second) and/or after having extrapolated the measured values to the point corresponding to the time-span extrapolated to zero. Non-compliance with the above measures gives rise to measured apparent ka values that are between one and two orders of magnitude too low. Potentially the greatest source of error is an instrumental factor (involving “inert” carrier surfaces) which, although unsuspected by most users, causes an often enormous, usually mainly non-electrostatic repulsion exerted between the dissolved hydrophilic biopolymeric ligand molecules and the equally hydrophilic material of the “inert” carrier of the embedded or attached receptor sites with all immersed in water. This extraneous, aspecific, macroscopic-scale hydrophilic repulsion gives rise to observed ka values which are 102 to 106 times too low. However, by means of surface-thermodynamic analyses of the surfaces of the biopolymeric ligands and of the receptor molecules, as well as of the carrier material, and subsequent extended DLVO analysis of the free energies thus obtained, the value for the specific microscopic-scale kinetic association rate constant, kamk may be isolated using von Smoluchowski’s approach. This value, in conjunction with Kaff (t ® 0), can then yield kdmk (t ® 0). The latter is the microscopic-scale dissociation rate constant which, contrary to kamk varies considerably from one system to another. It is this value that best characterizes the kinetic aspect of specific as well as of adsorption interactions.