Proton and Deuteron Nuclear Magnetic Relaxation Dispersion Studies of Ca²⁺-Mn²⁺-Concanavalin A: Evidence for Two Classes of Exchanging Water Molecules

We have measured the magnetic field dependence of the nuclear magnetic relaxation rates (NMRD profiles) of solvent protons and deuterons in solutions of Ca²⁺-Mn²⁺-concanavalin A (Con A) with and without saccharide present. Data were obtained over the range -8 to 35 °C; the extension to the lowest temperature was made possible by the presence of 5 M salt. Since previous theoretical analyses, using accepted relaxation theories of 1H NMRD profiles alone, led to unsatisfactory conclusions, we have attempted to take advantage of the fact that the residence lifetime of a water ligand of the metal ions can influence the relaxation behavior of protons and deuterons differently. From a comparison of the present proton and deuteron results, we find that Ca²⁺-Mn²⁺-Con A has two classes of binding sites: one, associated with the inner coordiation sphere of the Mn2+ ions, having a resident lifetime for solvent water of ~ 10~5 s that is reduced by the presence of saccharide and another having a lifetime of ~5 × 10-9 s, located with the protons of the bound waters ~4.4 Å from the Mn2+ ions (assuming two equivalent water molecules in this class), which is well beyond the coordination environment of the Mn²⁺ ions. The relaxation contribution of these more distant sites is unaffected by saccharide. The conclusions are corroborated by measurements of the temperature dependences of the proton NMRD profiles, which show quite clearly that the profiles are composite, containing two contributions with opposite dependences on temperature. The more slowly exchanging water molecules dominate proton relaxation above about 25 °C and dominate deuteron relaxation throughout. The more rapidly exchanging water molecules dominate proton relaxation at lower temperatures but make no detectable contribution to the deuteron data under these conditions. These results are the first in which it can be said with some confidence that exchanging water molecules liganded in two disparate classes of sites, with different exchange rates and different contributions to the NMRD profiles, have been identified.