Kinetic Analysis of the Mass Transport Limited Interaction between the Tyrosine Kinase lck SH2 Domain and a Phosphorylated Peptide Studied by a New Cuvette-Based Surface Plasmon Resonance Instrument
✍ Scribed by Nico J. de Mol; Emke Plomp; Marcel J.E. Fischer; Rob Ruijtenbeek
- Publisher
- Elsevier Science
- Year
- 2000
- Tongue
- English
- Weight
- 140 KB
- Volume
- 279
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
We explored the use of a newly developed cuvettebased surface plasmon resonance (SPR) instrument (IBIS) to study peptide-protein interactions. We studied the interaction between the SH2 domain of lck and a phosphotyrosine peptide EPQY*EEIPIYL which was immobilized on a sensor chip. No indications for mass transport limitation (MTL) were observed when standard kinetic approaches were used. However, addition of competing peptide during dissociation revealed a high extent of rebinding. A dissociation rate constant (k d ) of 0.6 ؎ 0.1 s ؊1 was obtained in the presence of large amounts of peptide. A simple bimolecular binding model, applying second-order kinetics for the cuvette system, could not adequately describe the data. Fits were improved upon including a step in the model which describes diffusion of the SH2 domain from the bulk to the sensor, especially for a surface with high binding capacity. From experiments in glycerol-containing buffers, it appeared that the diffusion rate decreased with higher viscosity. It is demonstrated that MTL during association and dissociation can be described by the same diffusion rate. A binding constant (K D ) of 5.9 ؎ 0.8 nM was obtained from the SPR equilibrium signals by fitting to a Langmuir binding isotherm, with correction for loss of free analyte due to binding. An association rate constant k a of 1.1(؎0.2) ؋ 10 8 M ؊1 ⅐ s ؊1 was obtained from k d /K D . The values for k a and k d obtained in this way were 2-3 orders larger than that from standard kinetic analysis, ignoring MTL. We conclude that in a cuvette the extent of MTL is comparable to that in a flow system.