Off-resonance rotating frame spin-lattice NMR relaxation studies of phosphorus metabolite rotational diffusion in bovine lens homogenates

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Abstract
The rotational diffusion behavior of phosphorus metabolites present in calf lens cortical and nuclear homogenates was investigated by the NMR technique of 31P off-resonance rotating frame spin-lattice relaxation as a means of assessing the occurrence and extent of phosphorus metabolite-lens protein interactions. 31P NMR spectra of calf lens homogenates were obtained at 10 and 18.degree. C (below and above the cold cataract phase transition temperature, respectively) at 7.05 T. Effective rotational correlation time (.tau.0,eff) for the major phosphorus metabolites present in cortical and nuclear bovine calf lens homogenates were derived from nonlinear least-squares analysis of R vs .omega.e (spectral intensity ratio vs precessional frequency about the effective field) data with the assumption of isotropic reorientational motion. Intramolecular dipole-dipole (1H-31P, 31P-31P), chemical shift anisotropy (CSA), and solvent (water) translational intermolecular dipole-dipole (1H-31P) relaxation contributions were assumed in the analyses. In those cases where the limiting value of the spectral intensity ratio failed to reach unity at large offset frequency, a modified formalism incorporating chemical exchange mediated saturation transfer between two sites was used. Values of .tau.0,eff for phosphorus metabolites present in the cortex varied from a low of ca. 2 ns [L-.alpha.-glycerophosphocholine (GPC)] to a high of 12 ns (.alpha.-ATP) at 10.degree. C, whereas at 18.degree. C the range was from ca. 1 to 9 ns. For the nucleus the .tau.0,eff values ranged from ca. 3 ns (GPC) to 41 ns (Pi) at 10.degree. C; at 18.degree. C the corresponding values ranged from 4 to 39 ns. For PME (phosphomonoester; in lens the predominant metabolite is L-.alpha.-glycerol phosphate) at 18.degree. C evidence was obtained for binding and subsequent exchange with solidlike protein domains. The diversity in .tau.0,eff values for lenticular phosphorus metabolites is suggestive of differential binding to more slowly tumbling macromolecular species, most likely lens crystallin proteins. Corresponding measurement of T0,eff values for the mobile protein fraction present in calf lens cortical and nuclear homogenates at 10 and 18.degree. C, by 13C off-resonance rotating frame spin-lattice relaxation, provided average macromolecular correlation times that were assumed to represent the bound metabolite state. A fast-exchange model (on the T1 time scale), between free and bound forms, was employed in the analysis of the metabolite R vs .omega.3 curves to yield the fraction of free (unbound) metabolite (.THETA.free). The following .THETA.free values for phosphorus metabolite in calf lens homogenates were obtained at 10 and 18.degree. C, respectively: (cortex) GPC, 0.98, 0.98; PME, 0.97, 0.92; Pi, 0.99, 0.99; ATP, 0.75, 0.85; (nucleus) GPC, 0.98, 0.96; PME, 0.80, 0.63; Pi 0.84, 0.87. The results of this study establish the occurrence of significant temperature-dependent (above and below the cold cataract phase transition temperature) binding of ATP (cortex) and PME (nucleus) and Pi (nucleus) in calf lens.

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