Expanding the range of free calcium regulation in biological solutions

David Dweck, Avelino Reyes-Alfonso, James D. Potter

Research output: Contribution to journalArticle

60 Scopus citations

Abstract

Many biological systems use ethylene glycol bis (β-aminoethylether)-N, N,N′,N′-tetraacetic acid (EGTA) to regulate the free calcium concentration ([Ca2+]free) in the presence of physiological levels of free Mg2+ ([Mg2+]free). Frequently, it is necessary to work at [Ca2+]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 Ca2+-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 Ca2+, ATP, and Mg 2+. The program is intuitive, user-friendly, and flexible enough to fix or vary the [Mg-ATP2-] 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.

Original languageEnglish (US)
Pages (from-to)303-315
Number of pages13
JournalAnalytical Biochemistry
Volume347
Issue number2
DOIs
StatePublished - Dec 15 2005

Keywords

  • Calcium binding
  • Calcium binding affinity
  • Calcium buffering
  • Calcium regulation
  • Calcium titration
  • Calculator program
  • Cation chelator
  • Chelator
  • EGTA
  • Free calcium
  • Free calcium concentration
  • Nitrilotriacetic acid
  • NTA
  • pCa
  • Troponin C

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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