Expanding the range of free calcium regulation in biological solutions |
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Authors: | Dweck David Reyes-Alfonso Avelino Potter James D |
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Affiliation: | Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami, Miami, FL 33136, USA. |
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Abstract: | Many biological systems use ethylene glycol bis (beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) to regulate the free calcium concentration ([Ca(2+)](free)) in the presence of physiological levels of free Mg(2+) ([Mg(2+)](free)). Frequently, it is necessary to work at [Ca(2+)](free) beyond EGTA's buffering capabilities. Therefore, we have developed methods to extend the buffering range by adding nitrilotriacetic acid (NTA) to solutions containing EGTA. This extension results from NTA having a lower K'(dCa) than EGTA. Such equilibria are solved by pCa Calculator, a computer program designed to aid in the study of Ca(2+)-dependent physiological processes while accounting for the effects of pH, temperature, and ionic strength. With multiple chelators and pH buffers from which to choose, pCa Calculator calculates the total concentration of each species required to achieve specified free concentrations of Ca(2+), ATP, and Mg(2+). The program is intuitive, user-friendly, and flexible enough to fix or vary the [Mg-ATP(2-)] and ionic strength. Moreover, it can account for increases in experimental volume from calcium addition. A comparative analysis is reported for testing solutions in the presence and absence of NTA by measuring the calcium binding affinity of fluorescent cardiac troponin C. These findings demonstrate that EGTA, when used in conjunction with NTA, improves and expands the regulation of free calcium in solution. |
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Keywords: | Calcium regulation Calcium buffering Calcium binding Calcium binding affinity Calcium titration Calculator program Calcium titration Free calcium Free calcium concentration pCa Cation chelator Chelator EGTA NTA Nitrilotriacetic acid Troponin C Magnesium Mg-ATP |
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