ISSN:
1398-9995
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Medicine
Notes:
Background: Several studies showed that calcium-binding proteins have a fixed place in the spectrum of allergenic substances. Often the binding of a calcium ion induces conformational changes and affects immunoglobulin E-binding to the allergen. Hence, the quantitative characterization of the binding to calcium is of importance to understand both the biologic and allergenic activity of these proteins.Aims of the study: In the present study we describe a procedure for determining the stoichiometry and dissociation constant (KD) of calcium-binding allergens using circular dichroism (CD) techniques. For the experiments, we used recombinant Bet v 4, a two EF-hand allergen from birch pollen.Methods: Solutions of Bet v 4 were titrated with calcium and the change in molar ellipticity at 222 nm was monitored with a CD spectropolarimeter.Results: The determination of the binding stoichiometry as well as of the KD for one EF-hand (4 μM) demonstrated the applicability of the method.Conclusions: CD-monitored calcium-titration of protein solutions represents a fast and easy method for determining the binding characteristics of calcium-binding allergens.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1111/j.1398-9995.2005.00907.x
