title: Structural changes in calcium-binding allergens: use of circular dichroism to study binding characteristics.
abstract: 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.
  
  
  OBJECTIVE
  In the present study we describe a procedure for determining the stoichiometry and dissociation constant (K(D)) 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 K(D) for one EF-hand (4 microM) 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.
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: D
authors.initials: D
authors.lastname: Hebenstreit
authors.forename: F
authors.initials: F
authors.lastname: Ferreira
mesh_terms: D000485:Allergens / Q000378:metabolism*; D052179:Antigens, Plant; D001665:Binding Sites / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002942:Circular Dichroism / Q000379:methods*; D010940:Plant Proteins / Q000378:metabolism*; D011058:Pollen / Q000378:metabolism*
chemical_list: D000485:Allergens; D052179:Antigens, Plant; C108867:BETV4 protein, Betula pendula; D002135:Calcium-Binding Proteins; D010940:Plant Proteins; D002118:Calcium