Stoichiometry of Manganese and Calcium Ion Binding to Concanavalin A

Using measurements of solvent nuclear (proton) magnetic relaxation dispersion (NMRD), we have previously shown that concanavalin A (Con A) can exist in two conformational forms and that, in the absence of Ca²⁺, Mn²⁺ can bind to both the S1 and S2 sites of each monomer of Con A of at least one conformer [Brown, R. D., III, Brewer, C. F., & Koenig, S. H. (1977) Biochemistry 16, 3883–3896]. Recently other investigators have claimed that the stoichiometry of Mn²⁺ binding to Con A is only 1:1 for this conformational state, both in the absence and presence of saccharide; the same was claimed for Ca²⁺ under similar conditions. We now present titration and equilibrium dialysis experiments, both in the absence and presence of saccharide, using NMRD and atomic absorption spectroscopy, to investigate the stoichiometry of Mn²⁺ and Ca²⁺ binding to Con A. We have extended the NMRD method to include the determination of the total concentration of Mn²⁺ in samples of Con A. This, coupled with our previous use of NMRD to measure the concentration of free Mn²⁺ in protein solutions as well as the distribution of bound Mn²⁺ among different sites, allows us to measure the stoichiometry of binding with precision. We reconfirm that, at equilibrium in the presence of excess Mn²⁺, the binding stoichiometry of Mn²⁺ to Con A is 2:1, both in the absence and presence of saccharide. Addition of Ca²⁺ to a solution of Mn²⁺-Con A results in stoichiometric displacement of Mn²⁺ from the S2 site under the conditions investigated. Under nonequilibrium conditions, Mn²⁺ forms a metastable binary complex with the protein that persists for days at 5 °C. We also report, for the first time, values for all of the dissociation constants of binary and ternary complexes of Mn²⁺ with both conformations of Con A in solution. Atomic absorption measurements also indicate that Ca²⁺, in the absence of Mn²⁺, binds to both S1 and S2 sites in the absence and presence of saccharides.