Kinetic analysis of the mass transport limited interaction between the tyrosine kinase Ick SH2 domain and a phosphorylated peptide studied by a new cuvette-based surface plasmon resonance instrument

被引:46
作者
de Mol, NJ [1 ]
Plomp, E [1 ]
Fischer, MJE [1 ]
Ruijtenbeek, R [1 ]
机构
[1] Univ Utrecht, Fac Pharm, Dept Med Chem, Utrecht Inst Pharmaceut Sci, NL-3508 TB Utrecht, Netherlands
关键词
D O I
10.1006/abio.1999.4464
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
We explored the use of a newly developed cuvette-based 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) x 10(8) M-1 . s(-1) was obtained from k(d)/K-D. The values for k(a) and k(d) obtained in this way were 23 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, (C) 2000 Academic Press.
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页码:61 / 70
页数:10
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