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 language | English |
|---|---|
| Pages (from-to) | 303-315 |
| Number of pages | 13 |
| Journal | Analytical Biochemistry |
| Volume | 347 |
| Issue number | 2 |
| DOIs | |
| State | Published - Dec 15 2005 |
| Externally published | Yes |
Fingerprint
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
Cite this
Expanding the range of free calcium regulation in biological solutions. / Dweck, David; Reyes-Alfonso, Avelino; Potter, James D.
In: Analytical Biochemistry, Vol. 347, No. 2, 15.12.2005, p. 303-315.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Expanding the range of free calcium regulation in biological solutions
AU - Dweck, David
AU - Reyes-Alfonso, Avelino
AU - Potter, James D.
PY - 2005/12/15
Y1 - 2005/12/15
N2 - 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.
AB - 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.
KW - Calcium binding
KW - Calcium binding affinity
KW - Calcium buffering
KW - Calcium regulation
KW - Calcium titration
KW - Calculator program
KW - Cation chelator
KW - Chelator
KW - EGTA
KW - Free calcium
KW - Free calcium concentration
KW - Nitrilotriacetic acid
KW - NTA
KW - pCa
KW - Troponin C
UR - http://www.scopus.com/inward/record.url?scp=27944491141&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=27944491141&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2005.09.025
DO - 10.1016/j.ab.2005.09.025
M3 - Article
C2 - 16289079
AN - SCOPUS:27944491141
VL - 347
SP - 303
EP - 315
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -