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Multi-competition binding measurements of Gd3+ affinity to troponin C



Multi-competition binding measurements of Gd3+ affinity to troponin C



Biophysical Journal 86(1): 394a-395a



The popularity of using lanthanides as NMR/EPR/fluorescent analogs of divalent ions in Ca-binding proteins prompted us to re-evaluate the binding stoichiometry and affinity of Gd3+. We utilized competition between Ca2+, Gd3+, the fluorescent Ca2+ indicators with varying Ca2+ affinity (Fluo-3, Fura-6F and Fluo-5N) and the cardiac troponin C (TnC) to investigate the high and low affinity sites and non-specific binding. The above fluorescence Ca2+ indicators are also sensitive to Gd3+. Cardiac TnC has three Ca2+ binding sites, two high affinity sites in C domain (site III/IV) and one low affinity site in N Domain (Site II). Gd is known to have higher affinity for Ca2+ binding sites than calcium and there is a chance of non-specific binding (B. Sykes, personal communication). First, we have confirmed the binding affinities of Ca2+ to site III/IV and site II using the competition method and found it to be in a good agreement with reported values. Affinity of Gd3+ was found to be 140 times stronger than of Ca2+ for high affinity sites (Kd=0.36 nM) and apprx70 times stronger for low affinity sites (29 nM). Additionally, we found two very low affinity Gd binding sites with Kd of 1.1 muM. The implications of these findings on spectroscopic measurements using Gd3+ as a probe will be discussed.

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Accession: 035349058

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