Determination of receptor-ligand kinetic and equilibrium binding constants using surface plasmon resonance: application to the lck SH2 domain and phosphotyrosyl peptides

J Med Chem. 1995 Apr 14;38(8):1309-18. doi: 10.1021/jm00008a009.


Experimental and computational methods were developed for surface plasmon resonance (SPR) measurements involving interactions between a solution-binding component and a surface-immobilized ligand. These protocols were used to distinguish differences in affinity between the SH2 domain of lck and phosphotyrosyl peptides. The surface-immobilized ligand was the phosphotyrosyl peptide EPQpYEEIPIA, which contains a consensus sequence (pYEEI) for binding lck SH2. In the kinetic experiment, SPR phenomena were measured during association and dissociation reactions for a series of glutathione-S-transferase (GST)-SH2 concentrations, generating a set of SPR curves. A global computational analysis using an A + B<==>AB model resulted in single set of parameter estimates and statistics. In an abbreviated format, an equilibrium experiment was designed so that equilibrium constants (Keq) could be determined rapidly and accurately. A competitive equilibrium assay was developed for GST-SH2 in which Keq values for a series of phosphotyrosyl peptides (derived from the pYEEI sequence) varied over 3 orders of magnitude. Interestingly, these results highlighted the significance of the +1 glutamate in providing high-affinity binding to the SH2 domain. For most drug discovery programs, these Keq determinations are a sufficient measure of potency for the primary screen, with koff and kon determined in a secondary assay. Thus, the application of these techniques to SPR binding phenomena should prove valuable in the discovery and design of receptor-ligand antagonists.

MeSH terms

  • Amino Acid Sequence
  • Binding, Competitive
  • Kinetics
  • Ligands
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck)
  • Molecular Sequence Data
  • Peptides / chemistry
  • Peptides / metabolism*
  • Phosphotyrosine
  • Protein Binding
  • Protein-Tyrosine Kinases / chemistry
  • Protein-Tyrosine Kinases / metabolism*
  • Spectrum Analysis / methods
  • Tyrosine / analogs & derivatives*
  • Tyrosine / chemistry
  • Tyrosine / metabolism


  • Ligands
  • Peptides
  • Phosphotyrosine
  • Tyrosine
  • Protein-Tyrosine Kinases
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck)