Measuring Ca2+ binding to short chain fatty acids and gluconate with a Ca2+ electrode: Role of the reference electrode

Many organic anions bind free Ca2+, the total concentration of which must be adjusted in experimental solutions. Because published values for the apparent dissociation constant (Kapp) describing the Ca2+ affinity of short chain fatty acids (SCFAs) and gluconate are highly variable, Ca2+ electrodes coupled to either a 3 M KCl or a Na+ selective electrode were used to redetermine Kapp. All solutions contained 130 mM Na+, whereas the concentration of the studied anion was varied from 15 to 120 mM, replacing Cl− that was decreased concomitantly to maintain osmolarity. This induces changes in the liquid junction potential (LJP) at the 3 M 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 5 mV 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 Ca2+ and that the Kapp for the binding of Ca2+ to gluconate at pH 7.4, ionic strength 0.15 M, and 23 °C was 52.7 mM.