Measuring Ca(2+) binding to short chain fatty acids and gluconate with a Ca(2+) electrode: role of the reference...

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.