Measuring Ca(2+) binding to short chain fatty acids and gluconate with a Ca(2+) electrode: role of the reference electrode.
Many organic anions bind free Ca(2+), the total concentration of which must be adjusted in experimental solutions. Because published values for the apparent dissociation constant (Kapp) describing the Ca(2+) affinity of short chain fatty acids (SCFAs) and gluconate are highly variable, Ca(2+) electrodes coupled to either a 3M KCl or a Na(+) selective electrode were used to redetermine Kapp. All solutions contained 130mM Na(+), whereas the concentration of the studied anion was varied from 15 to 120mM, replacing Cl(-) that was decreased concomitantly to maintain osmolarity. This induces changes in the liquid junction potential (LJP) at the 3M KCl reference electrode, leading to a systematic underestimation of Kapp if left uncorrected. Because the Na(+) concentration in all solutions was constant, a Na(+) electrode was used to directly measure the changes in the LJP at the 3 M KCl reference, which were under 5mV but twice those predicted by the Henderson equation. Determination of Kapp either after correction for these LJP changes or via direct reference to a Na(+) electrode showed that SCFAs do not bind Ca(2+) and that the Kapp for the binding of Ca(2+) to gluconate at pH 7.4, ionic strength 0.15M, and 23°C was 52.7mM.
Institute for Veterinary Physiology, Department of Veterinary Medicine, Freie Universität Berlin, 14163 Berlin, Germany.
Friederike
F
Stumpff
Institute for Veterinary Physiology, Department of Veterinary Medicine, Freie Universität Berlin, 14163 Berlin, Germany. Electronic address: jasmcguigan@zedat.fu-berlin.de.
John A S
JA
McGuigan
D015374:Biosensing Techniques / Q000295:instrumentation* / Q000592:standards*; D002118:Calcium / Q000378:metabolism*; D002138:Calibration; D004566:Electrodes; D005227:Fatty Acids / Q000737:chemistry* / Q000378:metabolism*; D005942:Gluconates / Q000378:metabolism*; D009994:Osmolar Concentration; D012015:Reference Standards
D005227:Fatty Acids; D005942:Gluconates; C030691:gluconic acid; D002118:Calcium
Ca(2+) affinity for SCFA; Ca(2+) affinity for gluconate; Ca(2+) macroelectrodes; Liquid junction potential; Reference electrodes