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 tem perature without reducing agents. In a typical experiment, the T-ph of the protein solution (218 mg/ml) increases from 2.5 +/- 1 degrees C t o 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 h igher than that of pure gamma II. The average apparent hydrodynamic ra dius 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 dime r of gamma II crystallin. gamma IIH has a lower thiol content than gam ma II and is not formed in the presence of dithiothreitol. We conclude that gamma IIH is a thiol oxidation product of gamma II-crystallin an d is a dimer containing an intermolecular disulfide crosslink Thus, so me oxidative modifications of protein thiol groups lead to an increase in net attractive interactions between proteins. As a result, T-ph in creases and protein aggregates are formed. These two microscopic chang es produce the increased light scattering associated with lens opacifi cation.