Aspartate (Asp) mononegative ion binds calcium through both carboxylates in contrast to binding through only the side chain carboxylate for mononegative glutamate (Glu), as shown by density functional theory (DFT) calculations. A stronger binding was confirmed electrochemically for Asp compared to Glu. From temperature dependence of binding constant, 15–37 °C investigated for aqueous 0.16 M NaCl, a more negative ΔH⁰ of − 21 kJ·mol⁻¹ was found for Glu compared to ΔH⁰ = −17 kJ·mol⁻¹ for Asp, a difference confirmed by DFT calculations and qualitatively also by isothermal titration calorimetry. The stronger binding of calcium to Asp (K_ass,c = 5.3 M⁻¹ at 37 °C) compared to Glu (K_ass,c = 3.6 M⁻¹ at 37 °C) despite the less negative enthalpy of binding is accordingly an entropy effect due to ring formation in the complex for Asp.