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Biophysical Journal 86:1959-1968 (2004)
© 2004 The Biophysical Society

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

Gertjan Veldhuis, Erwin P. P. Vos, Jaap Broos, Bert Poolman and Ruud M. Scheek

Department of Biochemistry and Biophysical Chemistry, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, 9747 AG Groningen, The Netherlands

Correspondence: Address reprint requests to Ruud M. Scheek, Tel.: +31-50-3634328; Fax: +31-50-3634800; E-mail: scheek{at}chem.rug.nl.

Flow dialysis has found widespread use in determining the dissociation constant (KD) of a protein-ligand interaction or the amount of available binding sites (E0). This method has the potency to measure both these parameters in a single experiment and in this article a method to measure simultaneously the KD and E0 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 KD and E0. 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.




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