Journal
ANALYTICAL BIOCHEMISTRY
Volume 305, Issue 2, Pages 195-205Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1006/abio.2002.5661
Keywords
Ca2+ binding; spectroscopy; software; computer fitting
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The complexity of Ca2+ cell signaling is dependent on a plethoria of Ca2+-binding proteins that respond to signals in different ranges of Ca2+ concentrations. Since the function of these proteins is directly coupled to their Ca2+-binding properties, there is a need for accurately determined equilibrium Ca2+-binding constants. In this work we outline the experimental techniques available to determine Ca2+-binding constants in proteins, derive the models used to describe the binding, and present CaLigator, software for least-square fitting directly to the measured quantity. The use of the software is illustrated for Ca2+-binding data obtained for two deamidated forms of calbindin D-9k, either an isospartate-56 (beta form) or a normal Asp-56 (alpha form). Here, the Ca2+-binding properties of the two isoforms have been studied using the chelator method. The a form shows similar Ca2+-binding properties to the wild type while the beta form has lost both cooperativety and affinity. (C) 2002 Elsevier Science (USA).
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