Biophysical Characterization of Macromolecules

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The determination of kinetic rate constants and of equilibrium constants of biomolecular interactions using optical biosensors has frequently suffered from lack of reliable results, in particular for high-affinity interactions. We have previously identified transport limitation of the mobile reactant to the binding sites immobilized at the sensor surface as one of the possible underlying causes of error in the kinetic analysis of biosensor data. We have developed an experimental configuration that allows the direct measurement of equilibrium binding constants by a modification of the sample handling system of our surface plasmon resonance biosensor. We have modified the analyte supply, previously achieved through a single injection of limited volume into a finite microfluidics loop. In the new configuration, we use an external syringe pump for the continuous recycling of the sample within a closed loop, thereby providing a virtually unlimited contact time of the sample with its immobilized sites. This allows the attainment of thermodynamic equilibrium of the interaction, even for very slow binding rates and high affinity interactions, and enables the measurement of equilibrium binding constants in an equilibrium titration approach. While this technique avoids the need to interpret the binding kinetics and avoids the related artifacts, it also eliminates the need for chemical regeneration in between the conventional multiple kinetic binding-desorption cycles. This method was used in initial applications in the study of lysozyme antibody interactions in collaboration with Dr. Smith-Gill. In a project using the conventional optical biosensor configuration in collaboration with Dr. Padlan and the laboratory of Dr. Helm, a new method was developed for comparative analysis of kinetic rate constants, and applied to the binding of a series of IgE mutants to the IgE-receptor for the identification of structurally important residues. Collaborative studies using the surface plasmon resonance technique have been started with the laboratories of Dr. Fishman on the characterization of G-protein interactions, and with the Dr. Sharma on the quantification of the interactions of cyclin-dependent kinases. This is a continuation of Intramural Research Project Z01-RR-10485-01 BEI.