Studies on the solubilization of the water-insoluble fraction from human lens and cataract.
Studies were carried out comparing the ability of urea extraction and sonication to solubilize the water-insoluble (WI) protein fraction from human lens tissue. Sonication and urea extraction were able to solubilize greater than 80% of the insoluble protein whether whole lenses or lens nuclei were used. This was true for normal lens and +1 cataracts; however, only 60% solubilization was obtained with the WI fraction from more advanced cataracts. Equal aliquots of a WI fraction from both pooled normal and pooled cataract lens nuclei were solubilized with and without reducing agents. The addition of dithiothreitol (DTT) had no significant effect on solubilization of the normal lens WI fraction. DTT did increase the protein solubilized from the cataract WI fraction by 30% with urea extraction; however, no increase was seen with sonication. When sodium borohydride was used as the reducing agent, essentially the same results were obtained. The solubilized protein populations were identical by SDS-PAGE and amino acid analysis. The addition of reducing agents had no effect on the amino acid content of the solubilized proteins with the single exception of lysine. This amino acid was markedly decreased in the proteins extracted in the presence of 40 mM sodium borohydride, but not with DTT. These data suggest that the borohydride not only increased the amount of protein solubilized, but likely also stabilized glycated lysine residues during the acid hydrolysis. Therefore, sonication readily provides a soluble preparation of the WI proteins from normal and cataract lens nuclei without the need for denaturing agents, however, disulfide-linked and lysine modified crystallins were best solubilized with urea.
Ortwerth
B J
BJ
Mason Institute of Ophthalmology, University of Missouri, Columbia 65212.
Olesen
P R
PR
D000596:Amino Acids / Q000032:analysis; D002386:Cataract / Q000378:metabolism*; D002850:Chromatography, Gel; D003459:Crystallins / Q000032:analysis* / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism; D006801:Humans; D007908:Lens, Crystalline / Q000737:chemistry*; D012995:Solubility; D013010:Sonication; D014508:Urea / Q000378:metabolism
D000596:Amino Acids; D003459:Crystallins; D014508:Urea; D004229:Dithiothreitol