Using measurements of the magnetic-field dependence of the nuclear magnetic relaxation rate ( I / T l ) of solvent water protons over a wide range of field values (corresponding to proton Larmor frequencies from 0.01 to 50 MHz), we have investigated the interaction of Mn2+ and Ca2+ ions with concanavalin A (Con A) over the pH range 5.3 to 6.4, at 5 and 25 "C. Particular attention was given to time-dependent effects that occur upon addition or removal of metals. Limited amounts of Mn2+ added to solutions of apo-Con A bind at SI (the usual "transition-metal"site) to form a binary complex characterized by a large and pH-dependent dissociation constant, rapid exchange of Mn2+ ions with solvent, and a relatively large and pH-independent contribution to the proton relaxation rate. With SI occupied, Ca2+ ions can bind at S2 (the usual "calcium-binding'' site) to form a metastable ternary complex characterized by a relatively large and pHdependent dissociation constant for Ca2+ ions, rapid exchange of Ca2+ ions with solvent, and a relatively low and pH-independent contribution to the proton relaxation rate. We find that this metastable ternary complex undergoes a first-order transition to a stable ternary complex, with a pH-independent time constant of 17 f 1 min at 5 " C and an activation energy of 22 kcal M-I. This stable ternary complex has the same relaxation contribution as the initial metastable complex, but differs in that the dissociation constant of Ca2+ is very low; the off-rate of both metals is of the order of days at 25 OC. Saccharide binding and agglutination studies are generally done with this form of Con A. We have also found that, in the abc o n c a n a v a l i n A, a metallo-protein isolated from the jack bean (Canavalia ensijormis), is one of a number of plant lectins. These proteins agglutinate cells in suspension with a selectivity that relates to the ability of lectins to bind to specific saccharides on cell surfaces (cf. Sharon and Lis, 1972; Lis and Sharon, 1973, for reviews). Interest in Con AI in particular arises from additional biological effects associated with the +From the IBM Thomas A ternary complex, respectively, of one particular conformation (called "unlocked") of Con A; PL, MPL, MMPL, and CMPL, the analogous forms of another conformation (called "locked") of Con A; MPE and CMPE, the thermal equilibrium states of samples of apo-Con A to which Mn2+, and subsequently Ca2+, respectively, have been added; [PSI, [MPS], and [MMPS], the sums of the concentrations of the respective unlocked and locked complexes; M and C, Mn2+ and Ca2+ ions, respectively, free in solution; EDTA, ethylenediaminetetraacetic acid. sence of Ca2+, Mn2+ can bind at S2 as well as at SI (S2 was previously thought to bind only Ca2+ and Cd2+) to form a metastable ternary complex which, like the metastable Mn2+-Ca2+-Con A complex, undergoes a transition to a stable state, but with a time constant that is much larger than for the Ca2+-containing ternary complex. In contrast to the stable Ca2+-Mn2+-Con A complex, the stab...

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Protein in water solution increases magnetic relaxation rates of solvent nuclei to an extent that depends on magnetic field strength and molecular weight. Koenig and Schillinger (J. Biol. Chem. 244, 3283 (1969)) showed that a small fraction of the water molecules in the first hydration shell, bound irrotationally with a residence lifetime in the range 0.1 to 10 microseconds, would account for the phenomena. No experiments, as yet, have proven the existence of such long-lived waters, nor yielded a value for their lifetime. Analogous measurements on solutions of both denatured and cross-linked protein give data different from that of native protein, but much like results for tissue. By comparing proton and deuteron relaxation rates in solutions of native and cross-linked protein, it is possible to demonstrate the existence of these relatively long-lived waters; the data indicate that 1% of a monolayer of the waters of hydration of protein have lifetimes that cluster near 1 microsecond and, it is argued, are held in place by multiple hydrogen bonds. Assigning shorter lifetimes for waters held by fewer bonds, it is possible to develop a unified view of relaxation of water nuclei in protein solutions and in tissue, and to relate it to recent crystallographic data on hydrated protein.

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Paramagnetic ions have been used to alter the magnetic relaxation rates 1/T1 and 1/T2 of solvent water protons since the first observations of a proton resonance signal almost four decades ago. The earliest theories of relaxation indicated that the influence of solute paramagnetic ions on relaxation rates of solvent protons should depend both on the chemical environment of the solute ions and on the magnetic field strength. Much knowledge, both experimental and theoretical, has since been amassed regarding relaxation effects in solutions of hydrated ions (aquoions) and of complexes of these ions with macromolecules, mainly proteins. The phenomena are well understood at this point, though the understanding is more retrospective than predictive for ion-protein complexes. Nonetheless, from what is now known about homogeneous solutions, and from current reports on the introduction of paramagnetic ions into tissue to alter contrast in NMR images by affecting relaxation rates, it is clear that the solution results are particularly germane, and transferable, to tissue investigations. The main features of relaxation of solvent protons in the presence of solute paramagnetic ions, as hydrated aquoions and complexed with protein, are presented here, with attention to those ions most relevant to in vivo studies, and with emphasis on the influence of the magnetic field and the chemical environment of these ions on solvent proton relaxation rates.