Disulfide cross-linking of urea-insoluble proteins in rabbit lenses treated with hyperbaric oxygen

Abstract

In vivo exposure of human patients and experimental animals to hyperbaric O2 has been shown by other investigators to lead to opacification of the lens especially in the nuclear region. In the present study, cultured rabbit lenses were treated with hyperbaric O2 in order to investigate possible formation of disulfide-cross-linked proteins in the urea-insoluble fraction of lens cortex and nucleus. When lenses were treated with 100 atmospheres of 100% O2 for 24 hr. intermolecular disulfide-linked proteins formed in both the cortical and nuclear regions. Under these conditions the level of reduced glutathione and the activity of glyceraldehyde-3 phosphate dehydrogenase (G-3PD) were depleted by greater than 95% in both regions. The lenses were hazy in appearance but not opaque. Two-dimensional diagonal electrophoresis followed by immunoblotting indicated that the majority of the cross-linked proteins were beta- and gamma-crystallins. Also involved in the cross-linking was the enzyme G-3PD but not the main intrinsic membrane protein. MIP26 kDa. Treatment of the nuclear urea-insoluble fraction of O2-treated lenses with sodium borohydride showed a nearly fourfold increase in the level of protein disulfide compared to that present in the same fraction of either fresh lenses or N2-treated controls. It was determined that an increase of approximately one disulfide group per 10(5) Da molecular weight corresponded to cross-linking of nearly 20% of the urea-insoluble protein present in the O2-treated lenses. Experiments carried out at 8 atmospheres O2 were used to determine the region of the lens in which urea-insoluble disulfide first formed after exposure to O2. After 8 hr of treatment of lenses with 8 atmospheres O2 an increase in protein disulfide was observed in the urea-insoluble proteins of the lens nucleus but not of the cortex. Under these conditions, the level of glutathione had decreased by 62% in the nucleus compared to only 13% in the cortex. Increasing the culture time to 16 hr under 8 atmospheres O2 produced a further increase in protein disulfide in the nuclear region. The formation of a small amount of cross-linked protein in the cortex and a significantly greater decrease of G-3PD activity in the lens nucleus compared to the cortex. The overall results of the study demonstrate that exposure of lenses to hyperbaric O2 leads to disulfide-cross-linking of crystallins in the urea-insoluble fraction and that the initial formation of protein disulfide as well as the initial loss of glutathione occurs first in the lens nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)