Abstract
Optical spectroscopic techniques can be used to measure Ca2+-binding constants when the Ca2+-bound and free forms of the protein display a difference in, for example, the UV absorbance, CD or fluorescence spectrum, or fluorescence polarization. One may then start with the Ca2+-free form, titrate in Ca2+ stepwise, measure a spectrum or intensity at each step, and obtain the binding constants from computer fitting to the data. The best accuracy is achieved when the protein concentration is roughly the same as the dissociation constant (the inverse of the binding constant) such that there are significant populations of both bound and free forms at several titration points. This limits the useful range of such direct measurements to binding constants below 106 M-1 (KD> 1 μM), because of the practical difficulty of making buffers with less than 0.5-1 μM free Ca2+. For Ca2+-binding proteins with affinities of 106 M-1 and up, one has to rely on indirect measurements. One popular such approach uses around 1 mM ethylenediaminetetracetic acid (EDTA) or ethylene glycol-bis N,N,N′,N′-tetraacetic acid (EGTA), and a much smaller amount of protein so that the free-Ca2+concentration is essentially controlled by the Ca2+-buffering capacity of EDTA or EGTA. A potential risk with such approaches is binding of EDTA or EGTA to the protein with consequences for its Ca2+affinity. Another type of indirect approach outlined in this chapter involves the use of a chelator whose absorbance or fluorescence is Ca2+dependent (1-3).
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Linse, S. (2002). Calcium Binding to Proteins Studied via Competition with Chromophoric Chelators. In: Vogel, H.J. (eds) Calcium-Binding Protein Protocols: Volume 2: Methods and Techniques. Methods in Molecular Biology™, vol 173. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-184-1:015
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DOI: https://doi.org/10.1385/1-59259-184-1:015
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