Lens ␣-crystallin is a 600 -800-kDa heterogeneous oligomer protein consisting of two subunits, ␣A and ␣B. The homogeneous oligomers (␣A-and ␣B-crystallins) have been prepared by recombinant DNA technology and shown to differ in the following biophysical/biochemical properties: hydrophobicity, chaperone-like activity, subunit exchange rate, and thermal stability. In this study, we studied their thermodynamic stability by unfolding in guanidine hydrochloride. The unfolding was probed by three spectroscopic parameters: absorbance at 235 nm, Trp fluorescence intensity at 320 nm, and far-UV circular dichroism at 223 nm. Global analysis indicated that a three-state model better describes the unfolding behavior than a two-state model, an indication that there are stable intermediates for both ␣A-and ␣B-crystallins. In terms of standard free energy (⌬G NU H2O ), ␣A-crystallin is slightly more stable than ␣B-crystallin. The significance of the intermediates may be related to the functioning of ␣-crystallins as chaperonelike molecules.Human lens proteins become progressively less soluble with age and cataract formation. The accumulation of high molecular mass aggregated and insoluble proteins was thought to be the main cause of lens opacity (1, 2). The major crystallin in high molecular mass and insoluble proteins, ␣-crystallin, has been thought to play a major role in the maintenance of lens transparency (3), as manifested by the recent finding that ␣-crystallin acts like a chaperone in in vitro experiments (4). However, the mechanisms of high molecular mass aggregation and insolubilization have not been well established. A reasonable assumption is that these events are related to proteins being in an unstable or not fully folded state, and thus tending to aggregate when their concentrations are high. A study on thermodynamic and kinetic stability may provide a reliable clue.An earlier report indicated that the native ␣-crystallin is thermally very stable (5). It does not denature even at 100°C, but undergoes a thermal transition at 60 -65°C. Later, it was reported that ␣-crystallin becomes partially unfolded at this temperature (6). The refolding is irreversible after exposure to the high temperature (6, 7). On the other hand, the reversibility of denaturant unfolding of ␣-crystallin has been reported (8, 9). The thermodynamic and kinetic stability, however, has not been studied in detail, presumably because of the heterogeneity and oligomeric nature of ␣-crystallin. In contrast, the monomeric ␥-crystallin has been extensively studied (10 -13). Recently, we reported a thermodynamic and kinetic study on ␥F-crystallin (14), which is the most stable ␥-crystallin gene product (15,16).To obtain pure homogeneous ␣-crystallin, we have cloned human lens ␣A-and ␣B-crystallins (17), which are good models for studying the unfolding and refolding properties of ␣-crystallin. In addition to the unfolding intermediate, we also need to consider whether ␣-crystallin has a dissociation intermediate (18). In this work, we used the chemica...

A Trp-free K KA-crystallin mutant (W9F) was prepared by site-directed mutation. This mutant appears to be identical to the wild-type in terms of conformation (secondary and tertiary structures). W9F was labeled with a sulfhydryl-specific fluorescent probe, 2-(4P-maleimidylanilino) naphthalene-6-sulfonate (MIANS), and used in a subunit exchange between K KA-and K KAcrystallins as well as between K KA-and K KB-crystallins, studied by measurement of fluorescence resonance energy transfer. Energy transfer was observed between Trp (donor, with emission maximum at 336 nm) of wild-type K KA-or K KB-crystallin and MIANS (acceptor, with absorption maximum at 313 nm) of labeled W9F when subunit exchange occurred. Time-dependent decrease of Trp and increase of MIANS fluorescence were recorded. The exchange was faster at 37³C than at 25³C. The energy transfer efficiency was greater between homogeneous subunits (K KA-K KA) than between heterogeneous subunits (K KA-K KB). A previous exchange study with isoelectric focusing indicated a complete but slow exchange between K KA and K KB subunits. The present study showed that the exchange was a fast process, and the different energy transfer efficiencies between K KA-K KA and K KA-K KB indicated that K KA-and K KB-crystallins were not necessarily structurally equivalent.z 1998 Federation of European Biochemical Societies.

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