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...
