Ortwerth B J, Olesen P R
Mason Institute of Ophthalmology, University of Missouri, Columbia 65212.
Exp Eye Res. 1992 Dec;55(6):777-83. doi: 10.1016/0014-4835(92)90004-c.
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.
开展了多项研究，比较尿素提取法和超声处理法溶解人晶状体组织中不溶于水（WI）蛋白质组分的能力。无论使用完整晶状体还是晶状体核，超声处理和尿素提取法均能溶解超过80%的不溶性蛋白质。正常晶状体和+1期白内障均是如此；然而，对于更晚期的白内障，WI组分的溶解率仅为60%。将来自正常晶状体核和白内障晶状体核混合样本的等量WI组分在有或没有还原剂的情况下进行溶解。添加二硫苏糖醇（DTT）对正常晶状体WI组分的溶解没有显著影响。对于尿素提取法，DTT确实使从白内障WI组分中溶解的蛋白质增加了30%；然而，超声处理法未观察到增加。当使用硼氢化钠作为还原剂时，得到了基本相同的结果。通过SDS-PAGE和氨基酸分析，溶解的蛋白质群体是相同的。除赖氨酸外，添加还原剂对溶解蛋白质的氨基酸含量没有影响。在40 mM硼氢化钠存在下提取的蛋白质中，这种氨基酸明显减少，但DTT处理时没有减少。这些数据表明，硼氢化钠不仅增加了溶解的蛋白质数量，而且可能在酸水解过程中稳定了糖化赖氨酸残基。因此，超声处理很容易从正常和白内障晶状体核中提供WI蛋白质的可溶性制剂，而无需变性剂，然而，二硫键连接和赖氨酸修饰的晶状体蛋白用尿素溶解效果最佳。
