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19:16
The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis
Published on: March 17, 2010
Determining the affinity and stoichiometry of interactions between unmodified proteins in solution using Biacore
Eric S Day1, Allan D Capili, Christopher W Borysenko
1Biogen Idec, Cambridge, MA 02142, USA. estevenday@gmail.com
Analytical Biochemistry
|May 29, 2013
Summary
This study introduces a novel Biacore method to accurately measure protein-ligand binding affinities and stoichiometries in solution without modifying the molecules. The technique is validated for analyzing interactions involving multivalent antibodies, offering a robust tool for biochemical research.
Area of Science:
- Biochemistry
- Biophysics
- Analytical Chemistry
Background:
- Accurate measurement of equilibrium binding affinities and stoichiometries is crucial for understanding molecular interactions.
- Conventional methods may require modification of binding partners, potentially altering their properties.
- Analyzing multivalent interactions can be complex with standard techniques.
Purpose of the Study:
- To develop and validate a general Biacore method for determining equilibrium binding affinities and stoichiometries of protein-ligand interactions in solution.
- To provide a method that avoids covalent modification of binding partners.
- To enable quantitative analysis of complex interactions, including those involving multivalency.
Main Methods:
- A Biacore method analyzing pre-equilibrated protein-ligand mixtures in solution.
- Utilizing a sensor chip surface that detects unbound analyte.
- Performing analysis under mass transport limited conditions to determine unbound analyte concentration from initial binding velocity.
- Fitting initial velocity versus varied binding partner concentration plots to a quadratic binding equation.
Main Results:
- The method successfully measured equilibrium binding affinities and stoichiometries for soluble Her2 extracellular domain with monovalent, bivalent, and trivalent anti-Her2 antibodies.
- Measured affinities agreed with conventional Biacore kinetic analysis.
- Stoichiometries of 1:1, 1:2, and 1:3 complexes were confirmed by gel filtration with in-line light scattering.
- The method is applicable for affinities ranging from 100 pM to 1 μM.
Conclusions:
- This general Biacore method provides a reliable approach for measuring binding affinities and stoichiometries of unmodified protein-ligand interactions in solution.
- It is particularly valuable for studying interactions where molecular modification is undesirable or where multivalency complicates analysis.
- The technique offers a robust alternative for quantitative binding studies in various biochemical and biophysical research areas.
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