SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Probing the Functional Heterogeneity of Surface Binding Sites by Analysis of Experimental Binding Traces and The Effect of Mass Transport Limitation
Juraj Svitel 1, Hacene Boukari 1, Donald Van Ryk 1, Richard C Willson 2 and Peter Schuck 3*
1 NIH
2 University of Houston
3 National Institutes of Health
* To whom correspondence should be addressed. E-mail: pschuck{at}helix.nih.gov.
Submitted on August 2, 2006
Revised on September 27, 2006
Accepted on 16 November 2006
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Abstract |
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Many techniques rely on the binding activity of surface immobilized proteins, including antibody based affinity biosensors for the detection of analytes, immunoassays, protein arrays, and surface plasmon resonance biosensors for the study of thermodynamic and kinetic aspects of protein interactions. In order to study the functional homogeneity of the surface sites and to characterize their binding properties, we have recently proposed a computational tool to determine the distribution of affinity and kinetic rate constants from surface binding progress curves (Biophys. J. 84 (2003) 4062-4077). It is based on modeling the experimentally measured binding signal as a superposition of signals from binding to sites spanning a range of rate and equilibrium constants, with regularization providing the most parsimonious distribution consistent with the data. In the present work, we have expanded the scope of this approach to include a compartment-like transport step, which can describe competitive binding to different surface sites in a zone of depleted analyte close to the sensor surface. This novel approach addresses a major difficulty in the analysis of surface binding where both transport limitation as well as unknown surface site heterogeneity may be present. In addition to the kinetic binding parameters of the ensemble of surface sites, it can provide estimates for effective transport rate constants. Using antibody-antigen interactions as experimental model systems, we studied the effects of the immobilization matrix and of the analyte flow-rate on the effective transport rate constant. Both were experimentally observed to influence mass transport. The approximate description of mass transport by a compartment model becomes critical when applied to strongly transport controlled data, and we examined the limitations of this model. While in the presence of only moderate mass transport limitation the compartment model provides a good description, this approximation breaks down for strongly transport-limited surface binding. In the latter regime we report experimental evidence for the formation of gradients within the sensing volume of the evanescent field biosensor used.
Key Words:
adsorption, mass transport limitation, protein immobilization, protein interactions, surface binding kinetics, surface plasmon resonance
