Enlighten Publications

In this section

Ionized concentrations in Ca2+ and Mg2+ buffers must be measured, not calculated

McGuigan, J. A.S., Kay, J. W. and Elder, H. Y. (2020) Ionized concentrations in Ca2+ and Mg2+ buffers must be measured, not calculated. Experimental Physiology, 105(3), pp. 427-437. (doi: 10.1113/EP088345) (PMID:31758871)

Preview

Text
215904.pdf - Accepted Version
3MB

Abstract

Modelling intracellular regulation of Ca2+/Mg2+ is now an established part of physiology. However, the conclusions drawn from such studies depend on accurate knowledge of intracellular [Ca2+], ∆[Ca2+] and the pK′ values for the intracellular binding of Ca2+/Mg2+. Calculation of [Ca2+]/[Mg2+] in buffers is normal. The eight freely available programs all give different values for the [Ca2+]/[Mg2+] in the buffer solutions, varying by up to a factor of 4.3. As a result, concentrations must be measured. There are two methods to do this, both based on the ligand optimization method (LOM): (1) calibration solutions from 0.5 to 4 mmol l−1; and (2) calibration solutions from 0.1 µmol l−1 to 2 mmol l−1. Both methods can be used to calibrate Ca2+/Mg2+ electrodes. Only Method 2 can be used directly to calibrate fluorochromes and aequorin. Software in the statistical program R to calculate the [Ca2+]/[Mg2+] in buffers is provided for both methods. The LOM has now been generalized for use with electrodes, fluorochromes and aequorin, making it the ideal method to determine the pK′ values for intracellular binding of Ca2+/Mg2+. The [Ca2+]/[Mg2+] in buffers must be measured routinely, which is best done by calibrating electrodes with the LOM and software written in R. If [Ca2+]/[Mg2+] in buffers are calculated, the parameters used in modelling show the same degree of variability as the software programs. Uncritical acceptance of such parameters means that conclusions reached from such studies are relative, not absolute, and must now be re‐examined.

Item Type:	Articles
Keywords:	Ca2+ /Mg2+ buffers, Ca2+ /Mg2+ electrodes, ligand optimisation method, measurement of [Ca2+]/[Mg2+] in buffers.
Status:	Published
Refereed:	Yes
Glasgow Author(s) Enlighten ID:	Elder, Dr Hugh and Kay, Dr James
Authors:	McGuigan, J. A.S., Kay, J. W., and Elder, H. Y.
College/School:	College of Medical Veterinary and Life Sciences > School of Life Sciences College of Science and Engineering > School of Mathematics and Statistics > Statistics
Journal Name:	Experimental Physiology
Publisher:	Wiley
ISSN:	0958-0670
ISSN (Online):	1469-445X
Published Online:	23 November 2019
Copyright Holders:	Copyright © 2019 The Authors
First Published:	First published in Experimental Physiology 105(3): 427-437
Publisher Policy:	Reproduced in accordance with the publisher copyright policy

University Staff: Request a correction | Enlighten Editors: Update this record

Altmetric

References

Allen, D. G., Blinks, J. B., & Prendergast, F. G. (1977). Aequorin luminescence: Relation of light emission to calcium concentration—a calcium- independent component. Science, 195, 996–998. Bayliss, W. M. (1915). Principles of general physiology (1st edn, pp. 904). London: Longmans, Green & Co. Bers, D. M., Patton, C. W., & Nuccitelli, R. (1994). A practical guide to the preparation of Ca2+ buffers. Methods in Cell Biology, 40, 3–29. Blatter, L., & McGuigan, J. A. S. (1991). Intracellular pH regulation in ferret ventricular muscle. The role of Na–H exchange and the influence of metabolic substrates. Circulation Research, 68, 150–161. Blinks, J. R. (1989). Use of calcium-regulated photoproteins as intracellular Ca2+ indicators. Methods in Enzymology, 172, 164–203. Blinks, J. R., Wier, W. G., Hess, P., & Prendergast, F. G. (1982). Measurement of Ca2+ concentrations in living cells. Progress in Biophysics and Molecular Biology, 40, 1–114. Brooks, S. P. J., & Storey, K. B. (1992). Bound and determined: A computer program for making buffers of defined ion concentrations. Analytical Biochemistry, 201, 119–126. Clark, A. J. (1913). The actions of ions and lipoids upon frog’s heart. The Journal of Physiology, 47, 66–107. Dale, D. (1913). On the action of electrolytes on Paramoecium. The Journal of Physiology, 46, 129–140. Droogmans, G. (2005). CalBuf program. Retrieved from https://drive. google.com/open?id=1i7GU_yXSNSv-Tm6xToNSMiv5uxZCGe0i Ellis, D., & Thomas, R. C. (1976). Direct measurement of the intracellular pH of mammalian cardiac muscle. The Journal of Physiology, 262, 755–771. Fabiato, A., & Fabiato, F. (1979). Calculator programmes for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. Journal of Physiology (Paris), 75, 463–505. Fernbach, M. M. A., & Hubert, L. (1900). De l’influence de phosphates et de quelques autres matiéres minerals sur la diastase protéolytique du malt. Comptes rendus hebdomadaires des séances de l’Académie des sciences, 131, 293–295. Fry, C. H., & Langley, S. E. M. (2001). Ion-selective electrodes for biological systems (pp. 160). The Netherlands, Amsterdam: Hardwood Academic Publishers. Föhr, K. J., Warchol, W., & Gratzl, M. (1993). Calculation and control of free divalent cations in solutions used for membrane fusion studies. Methods in Enzymology, 221, 149–157. Godt, R. E., & Lindley, B. D. (1982). Influence of temperature upon contra- ctile activation and isometric force production in mechanically skinned fibres of the frog. Journal of General Physiology, 80, 279–297. Kay, J. W., Stevens, R., McGuigan, J. A. S., & Elder, H. Y. (2008). Auto- matic determination of ligand purity and apparent dissociation constant (Kapp) in Ca2+/Mg2+ buffer solutions and the Kapp for Ca2+/Mg2+ anion binding in physiological solutions from Ca2+/Mg2+-macroelectrode measurements. Computers in Biology and Medicine, 38, 101–110. Koppel, K., & Spiro, K. (1914). Ueber die Wirkung von Moderatoren (Puffern) bei der Verschiebung des Säure-Basengleichgewichtes in biologischen Flüssigkeiten. Biochemische Zeitschrift, 65, 409–439. Lüthi, D., Spichiger, U., Forster, I., & McGuigan, J. A. S. (1997). Calibration of Mg2+ -selective electrodes down to 1 μmol l−1 in intracellular and Ca2+ - containing extracellular solutions. Experimental Physiology, 82, 453–467. McGuigan, J. A. S., Kay, J. W., & Elder, H. Y. (2006). Critical review of the methods used to measure the apparent dissociation constant and ligand purity in Ca2+ and Mg2+ buffer solutions. Progress in Biophysics and Molecular Biology, 92, 333–370. McGuigan, J. A. S., Kay, J. W., & Elder, H. Y. (2014). An improvement to the ligand optimisation method (LOM) for measuring the apparent dissociation constant and ligand purity in Ca2+ and Mg2+ buffer solutions. Progress in Biophysics and Molecular Biology, 116, 203–211. McGuigan, J. A. S., Kay, J. W., & Elder, H. Y. (2017). Ionised concentrations in calcium and magnesium buffers: Standards and precise measurement are mandatory. Progress in Biophysics and Molecular Biology, 126, 48–64. McGuigan, J. A. S., Kay, J. W., Elder, H. Y., & Lüthi, D. (2007). Comparison between measured and calculated ionized concentrations in Mg2+ /ATP, Mg2+ /EDTA and Ca2+ /EGTA buffers; influence of changes in temperature, pH and pipetting errors on the ionized concentrations. Magnesium Research, 20, 72–81. McGuigan, J. A. S., & Stumpff, F. (2013). Calculated and measured [Ca2+] in buffers used to calibrate Ca2+ -macroelectrodes. Analytical Biochemistry, 436, 29–35. Miller, D. J., & Smith, G. L. (1984). EGTA purity and the buffering of calcium ions in physiological solutions. American Journal of Physiology-Cell Physio- logy, 246, C160–C166. Neumaier, F., Alpdogan, S., Hescheler, J., & Schneider, T. (2018). A practical guide to the preparation and use of metal ion-buffered systems for physiological research. Acta Physiologica, 222, e12988, https://doi.org/10.1111/apha.12988 R Core Team (2018). R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. Retrieved from https://www.R-project.org/ Reverdin, E., Illanes, A., & McGuigan, J. A. S. (1986). Internal potassium in ferret ventricle. Quarterly Journal of Experimental Physiology, 71, 451– 465. Ross, A., & Boron, W. F. (1980). The buffer value of weak acids and bases: Origin of the concept, and first mathematic derivation and application to physico-chemical systems. The work of M Koppel and K Spiro (1914). Respiration Physiology, 40, 1–32. Schoenmakers, T. J. M., Visser, G. J., Flik, G., & Theuvenet, A. R. P. (1992). Chelator: An improved method for computing metal ion concentrations in physiological solutions. Biotechniques, 12, 870–879. Sörensen, S. P. L. (1909). Enzymstudien. II. Ueber die Messung und die Bedeutung der Wasserstoffionenkonzentration bei enzymatischen Prozessen. Biochemische Zeitschrift, 21, 131–304. Stumpff, F., & McGuigan, J. A. S. (2014). Measuring Ca2+ binding to short chain fatty acids and gluconate with a Ca2+ electrode: Role of the reference electrode. Analytical Biochemistry, 459, 46–52. Tran, V., Parks, M. C. H., & Stricker, C. (2018). An improved measurement of the Ca2+ -binding affinity of fluorescent Ca2+ indicators. Cell Calcium, 71, 86–94. Tsien, R. Y., & Rink, T. J. (1980). Neutral carrier ion-selective microelectrodes for measurement of intracellular free calcium. Biochimica et Biophysica Acta, 839, 623–638. Weber, A., & Murray, J. M. (1973). Molecular control mechanisms in muscle contraction. Physiological Reviews, 53, 612–673. Woods, N. M., Cuthbertson, K. S. R., & Cobbold, P. H. (1986). Repetitive transient rises in cytoplasmic free calcium in hormone-stimulated hepatocytes. Nature, 319, 600–602. Zhang, W., Truttmann, A. C., Lüthi, D., & McGuigan, J. A. S. (1997). Apparent Mg2+ –adenosine 5-triphosphate dissociation constant measured with Mg2+ macroelectrodes under conditions pertinent to 31 P NMR ionized magnesium determinations. Analytical Biochemistry, 251, 246–250.

Deposit and Record Details

ID Code:	215904
Depositing User:	Dr James Kay
Datestamp:	15 May 2020 08:21
Last Modified:	25 Nov 2020 09:02
Date of acceptance:	22 November 2019
Date of first online publication:	23 November 2019
Date Deposited:	19 May 2020
Data Availability Statement:	No