Oxidation of γII-crystallin solutions yields dimers with a high phase separation temperature

Aqueous solutions of the bovine eye lens protein γII (or γB)-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°C to 32.5 ± 1°C after 21 days, and a new protein species, γII(H), is formed. The T(ph) of pure γII(H) is at least 40°C higher than that of pure γII. The average apparent hydrodynamic radius is 36 Å for γII(H) compared to 26 Å for γII. The molecular mass of γII(H) is ≃41.5 kDa compared to 20 kDa for native γII. Therefore, γII(H) is probably a dimer of γII crystallin. γII(H) has a lower thiol content than γII and is not formed in the presence of dithiothreitol. We conclude that γII(H) is a thiol oxidation product of γII-crystallin and is a dimer containing an inter-molecular 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.