Biophysical Journal
Volume 79, Issue 6, December 2000, Pages 3009-3018
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Binding Kinetics of Calbindin-D28k Determined by Flash Photolysis of Caged Ca2+

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Abstract

We have used UV flash photolysis of DM-nitrophen in combination with model-based analysis of Oregon Green 488 BAPTA-5N fluorescence transients to study the kinetics of Ca2+ binding to calbindin-D28K. The experiments used saturated DM-nitrophen at a [Ca2+] of 1.5 μM. Under these conditions, UV laser flashes produced rapid steplike increases in [Ca2+] in the absence of calbindin-D28K, and in its presence the decay of the flash-induced fluorescence was due solely to the Ca2+ buffering by the protein. We developed a novel method for kinetic parameter derivation and used the synthetic Ca2+ buffer EGTA to confirm its validity. We provide evidence that calbindin-D28K binds Ca2+ in at least two distinct kinetic patterns, one arising from high-affinity sites that bind Ca2+ with a kon comparable to that of EGTA (i.e., ∼1 × 107 M−1 s−1) and another with lower affinity and an approximately eightfold faster kon. In view of the inability of conventional approaches to adequately resolve rapid Ca2+ binding kinetics of Ca2+ buffers, this method promises to be highly valuable for studying the Ca2+ binding properties of other biologically important Ca2+ binding proteins.

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