Stoichiometry of magnesium and calcium ion binding to concanavalin A

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Abstract
Titration and equilibrium dialysis experiments were presented, in the absence and presence of saccharide, using NMRD [nuclear (proton) magnetic relaxation dispersion] and atomic absorption spectroscopy, to investigate the stoichiometry of Mn2+ and Ca2+ binding to ConA [concanavalin A, a metalloprotein isolated from the jack bean (Canavalia ensiformis)]. The NMRD method was extended to include the determination of the total concentration of Mn2+ in samples of Con A. This, coupled with the 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 measurement of the stoichiometry of binding with precision. 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.degree. C. Values were reported 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.