Evaluation of the Flow-Dialysis Technique for Analysis of Protein-Ligand Interactions: An Experimental and a Monte Carlo Study

Flow dialysis has found widespread use in determining the dissociation constant ( K D) of a protein-ligand interaction or the amount of available binding sites ( E 0). This method has the potency to measure both these parameters in a single experiment and in this article a method to measure simultan...

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Bibliographic Details
Published in:Biophysical journal 2004-04, Vol.86 (4), p.1959-1968
Main Authors: Veldhuis, Gertjan, Vos, Erwin P.P., Broos, Jaap, Poolman, Bert, Scheek, Ruud M.
Format: Article
Language:English
Subjects:
Binding sites
Biophysical Theory and Modeling
Biophysics
Cellular biology
Computer Simulation
Error analysis
Ligands
Measurement techniques
Microdialysis - methods
Monte Carlo Method
Protein Binding
Proteins
Proteins - chemistry
Simulation
Ultrafiltration
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Summary:Flow dialysis has found widespread use in determining the dissociation constant ( K D) of a protein-ligand interaction or the amount of available binding sites ( E 0). This method has the potency to measure both these parameters in a single experiment and in this article a method to measure simultaneously the K D and E 0 is presented, together with an extensive error analysis of the method. The flow-dialysis technique is experimentally simple to perform. However, a number of practical aspects of this method can have a large impact on the outcome of K D and E 0. We have investigated all sources of significant systematic and random errors, using the interaction between mannitol and its transporter from Escherichia coli as a model. Monte Carlo simulations were found to be an excellent tool to assess the impact of these errors on the binding parameters and to define the experimental conditions that allow their most accurate estimation.
ISSN:0006-3495
1542-0086
DOI:10.1016/S0006-3495(04)74259-9