OXIDATION OF GAMMA-II-CRYSTALLIN SOLUTIONS YIELDS DIMERS WITH A HIGH PHASE-SEPARATION TEMPERATURE

Aqueous solutions of the bovine eye lens protein gamma II (or gamma B)-crystallin at neutral pH show a gradual increase in phase separation temperature, T-ph, when allowed to stand for several weeks at room temperature without reducing agents. In a typical experiment, the T-ph of the protein solution (218 mg/ml) increases from 2.5 +/- 1 degrees C to 32.5 +/- 1 degrees C after 21 days, and a new protein species, gamma IIH, is formed. The T-ph of pure gamma IIH is at least 40 degrees C higher than that of pure gamma II. The average apparent hydrodynamic radius is 36 Angstrom for gamma IIH compared to 26 Angstrom for gamma II. The molecular mass of gamma IIH is approximate to 41.5 kDa compared to 20 kDa for native gamma II. Therefore, gamma IIH is probably a dimer of gamma II crystallin. gamma IIH has a lower thiol content than gamma II and is not formed in the presence of dithiothreitol. We conclude that gamma IIH is a thiol oxidation product of gamma II-crystallin and is a dimer containing an intermolecular disulfide crosslink Thus, some oxidative modifications of protein thiol groups lead to an increase in net attractive interactions between proteins. As a result, T-ph increases and protein aggregates are formed. These two microscopic changes produce the increased light scattering associated with lens opacification.