High molecular weight protein aggregates in X-ray-induced cataract

Abstract

The nature and the possible mechanism of formation of high molecular weight (HMW) protein aggregates were studied in X-ray-induced cataract in rabbit. The HMW protein fraction (molecular weight >4×10 6 daltons) which constituted approximately 18% of the total soluble protein in both the cataractous cortex and nucleus was isolated by gel filtration chromatography. The concentration of sulfhydryl (-SH) groups per milligram of protein in the HMW fraction was three times higher than that of normal α-crystallin. In addition, 50% of the total concentration of sulfhydryl groups contained in the HMW protein was found to be present in the oxidized state; the increased oxidation of protein -SH groups in the X-irradiated lens being observed only during the final stage of cataract which was marked by complete opacity. Treatment of HMW protein with dithioerythritol and subsequent refractionation yielded two peaks; peak I which eluted as α-crystallin and the second fraction (peak II) which eluted in the position of low molecular weight β-crystallin. Peak I accounted for approximately 40% of the deaggregated protein and peak II 60%. There was little protein eluted in the position of γ-crystallin. Thus, it appears that the HMW protein of X-ray-induced cataract is comprised of both α- and β-crystallins joined by intermolecular disulfide bonds. SDS (sodium dodecyl sulfate) electrophoresis of HMW protein and peaks I and II showed that the major bands of both peaks I and II were represented in the bands exhibited by the HMW protein. The electrophoretic mobilities of the major bands of peaks I and II were similar to those of α-crystallin and β-crystallin, respectively. The amino acid composition of peak I was found to be comparable to α-crystallin while the composition of peak II was similar to β-crystallin with the major exception that the hydrolysates of proteins in both peaks I and II were nearly lacking in tyrosine.