  Rebinding of dissociated ligands from cell surface proteins can confound
quantitative measurements of dissociation rates important for characterizing
the affinity of binding interactions. This can be true also for in vitro
techniques such as surface plasmon resonance (SPR). We present experimental
results using SPR for the interaction of insulin-like growth factor-I (IGF-I)
with one of its binding proteins, IGF binding protein-3 (IGFBP-3), and show
that rebinding, even with the addition of soluble heparin in the dissociation
phase, does not exhibit the expected exponential decay characteristic of a 1:1
binding reaction. We thus consider the effect of (multiple) rebinding events
and, within a self-consistent mean-field approximation, we derive the complete
mathematical form for the fraction of bound ligand as a function of time. We
show that, except for very low surface coverage/association rate, this function
is non-exponential at all times, indicating that multiple rebinding events
strongly influence dissociation even at early times. We compare the mean-field
results with numerical simulations and find good agreement, although deviations
are measurable in certain cases. Our analysis of the IGF-I-IGFBP-3 data
indicates that rebinding is prominent for this system and that the theoretical
predictions fit the experimental data well. Our results provide a means for
analyzing SPR biosensor data where rebinding is problematic and a methodology
to do so is presented.
