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

摘要

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 (K_(app)) describing the Ca~(2+) affinity of short chain fatty acids (SCFAs) and gluconate are highly variable, Ca~(2+) electrodes coupled to either a 3 M KCl or a Na~+ selective electrode were used to redetermine K_(app). 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 K_(app) 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 K_(app) 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 K_(app) for the binding of Ca~(2+) to gluconate at pH 7.4, ionic strength 0.15 M, and 23 ?C was 52.7 mM.