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M.F. Simpanya, R.R. Ansari, V.R. Leverenz, J.F. King, F.J. Giblin; Oxygen–Induced Aggregation of Lens Proteins in a Guinea Pig Model for Nuclear Cataract, Detected in vivo With Dynamic Light Scattering and in vitro With HPLC . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3881.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Treatment of guinea pigs with hyperbaric oxygen (HBO) is known to produce an increased level of lens nuclear light scattering (NLS). Here we used techniques of dynamic light scattering (DLS)and HPLC to study a possible link between increased NLS and aggregation of crystallins in the lens nucleus. Methods: Guinea pigs were treated 3x per week for 6 months with hyperbaric oxygen to increase the level of lens NLS. DLS in vivo was used to measure the sizes of lens proteins at 50 different locations across the optical axis of the guinea pig lens. Control and experimental lenses were divided into cortex and nucleus, homogenized, and the water insoluble (WI) protein pellets isolated. Care was taken to prevent artifactual production of protein disulfides. The WI protein pellets were dissolved in 6M guanidine/EDTA, and the proteins were separated by gel filtration HPLC, with and without reduction of disulfide, on a Biosep–SEC–S4000 column with a molecular weight range of 15–2,000 KDa. Pooled fractions of various peaks were dialyzed to remove guanidine, and then concentrated. Proteins present in various fractions were separated by SDS–PAGE, after reduction of –SS–, and analyzed by Western blots using antibodies to alpha, beta, gamma and zeta crystallins. Results: DLS data were analyzed for 4 control and 9 experimental animals. The mean protein size in the nucleus of lenses of O2–treated guinea pigs (216 measurements/9 lenses) was twice that of the controls (P<0.01). No significant size changes were observed for the cortex. A protein size distribution analysis was conducted at one point in the nucleus for control and experimental animals. One cluster of experimental nuclear proteins, accounting for 26% of the total, had a mean size 44x that of the major control group. HPLC analysis of guanidine–dissolved WI proteins, not reduced with DTT, showed a broad 150–1,000 KDa "shoulder" of aggregate protein, not present in the control. Reduction with DTT shifted all of this protein to a lower MW. All crystallins, except alpha–B (no –SH group content), were present in the aggregate protein, and all of the sample's zeta crystallin was in this HMW fraction. Conclusions: Large protein aggregates seen in vivo with DLS and in vitro with HPLC appear responsible for the increased NLS seen in the guinea pig/HBO experimental model. Disulfide is the major form of crosslinking observed. All the crystallins, except alpha–B, participate in the disulfide crosslinking, and zeta crystallin is particularly susceptible.
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