TY - JOUR
T1 - Probing the functional heterogeneity of surface binding sites by analysis of experimental binding traces and the effect of mass transport limitation
AU - Svitel, Juraj
AU - Boukari, Hacène
AU - Van Ryk, Donald
AU - Willson, Richard C.
AU - Schuck, Peter
N1 - Funding Information:
This work was supported in part by the Intramural Research Program of the National Institutes of Health. R.C.W. acknowledges support from the National Science Foundation and the Welch Foundation (grant No. E-1264).
PY - 2007/3
Y1 - 2007/3
N2 - 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. 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. 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 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. In the presence of only moderate mass transport limitation the compartment model provides a good description, but 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.
AB - 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. 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. 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 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. In the presence of only moderate mass transport limitation the compartment model provides a good description, but 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.
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U2 - 10.1529/biophysj.106.094615
DO - 10.1529/biophysj.106.094615
M3 - Article
C2 - 17158569
AN - SCOPUS:33847796944
SN - 0006-3495
VL - 92
SP - 1742
EP - 1758
JO - Biophysical Journal
JF - Biophysical Journal
IS - 5
ER -