Relaxometry of lens homogenates. II. temperature dependence and comparison with other proteins

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Abstract
We have extended our earlier work (C. F. Beaulieu, J. I. Clark, R. D. Brown III, M. Spiller, and S. H. Koenig, Magn. Reson. Med.8, 45 (1988)) on the magnetic field dependence of 1/T1 (NMRD profiles) of calf lens nuclear homogenates, at 25°C, to 5°C, and to other protein systems as well. These include concentrated solutions of myoglobin and bovine serum albumin, both globular proteins, the first compact and roughly spherical, the other extended, flexible, and with weak internal bonding; chicken lens homogenate, for which the dominant crystallins (lens proteins) are ∼ 70% α‐helical compared with calf crystallins, which are essentially all β‐sheet; and hen egg white, both native and heat‐ denatured. Our earlier conjectures regarding a reversible change in protein organization of the calf lens crystallins as a function of solute protein concentration is given added support. Our findings suggest that cytoplasmic homogenate can be characterized as a heterogeneous and polymorphic solution of crystallins. At high concentrations the NH moieties of the protein backbone become accessible to solvent with water (not NH proton) exchange rates > 104s−1. This conclusion is based on two aspects of the observed NMRD profiles. At low crystallin concentration, the profiles of calf and chicken lens homogenates are similar in form to those of myoglobin and native hen egg white, a form that has been studied previously for a range of diamagnetic globular proteins and has been demonstrated to arise from the rotational thermal motion of the solute molecules. At high crystallin concentrations, the NMRD profiles of the lens homogenates develop a monotonic background (high rates at low fields), much like that of the heat‐denatured egg‐white sample and those of most tissues. In addition, there is a set of peaks in the central part of the profiles of the concentrated crystallins, seen also in the denatured egg white and some tissues but not in the myoglobin sample, which is known to arise from cross‐relaxation interactions between the water protons and (through the intermediary of the NH proton) the 14N quadrupolar levels. The magnitude of these peaks, which is larger by an order of magnitude for native calf lens homogenates than for any tissue, requires that the majority of the NH moieties be accessible to water. Finally, going to 5°C for the native calf lens homogenate takes the sample below the temperature of reversible phase separation, and it becomes opaque. However, there is no change in the profile that can be attributed to this transition and its associated opacity. © 1989 Academic Press, Inc.