UV RADIATION CONFIGURATIONAL ALTERATIONS IN OCULAR LENS PROTEINS

晶状体蛋白的紫外线辐射构型变化

• 批准号: 6240319
• 负责人: LISA B HIBBARD
• 金额: $ 4.82万
• 依托单位: SPELMAN COLLEGE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6240319
• 关键词: SDS polyacrylamide gel electrophoresis; adenosinetriphosphatase; animal tissue; cataract; cations; crystallins; cyclic AMP; divalent cations; enzyme activity; enzyme structure; fluorescence spectrometry; glutathione reductase; glyceraldehyde 3 phosphate dehydrogenase; high performance liquid chromatography; human tissue; infrared spectrometry; ionic strengths; laboratory rat; photolysis; protein structure; radiation sensitivity; radiobiology; tryptophan; ultraviolet radiation; visual photosensitivity

It has been proposed by various investigators that UV radiation may be a causative factor in the formation of cataracts. Experimental models involving the structural proteins (crystallins) and enzymes of the ocular lens of various animal species have been used successfully to study the effects of near UV-A radiation both in vivo and in vitro. However, only recently has attention been given to UV-B radiation in the range of 300-33- nm. Little work has been performed to correlate UV-induced alterations in lens proteins and enzymes with changes such as increased Na+ and Ca2+ lens concentrations which are found in certain types of cataracts. The main objective of this research, thus, is to observe alterations in the structure of various ocular lens proteins and enzymes caused by near UN-B radiation plus UV-A radiation, and variaitons in ionic strength. These effects will be correlated with changes found in the structural proteins and enzymes of the ocular lens during cataract formation. To accomplish these investigations, solutions of lens crystallins or enzymes containing various electrolytes (NaCl, KCI, CaCI2, MgSO4) will be exposed to either 295 nm, 310 nm, or a combination of the two wavelengths from a 450 watt xenon lamp or 337.1 nm from a pulsed nitrogen laser and compared to non-irradiated control samples. Whole rat or bovine lenses will also be exposed to UV radiation after which the component enzymes will be isolated and analyzed. Photolysis of tryptophan residues, photoproduct formation, and alterations in configuration will be monitored using the spectroscopic techniques of fluorescence, absorbance, and Fourier transform infrared spectroscopy. Information obtained from previous steady-state fluorescence studies will be expanded to include lifetime and anisotropy data. The technique of SDS-polyacrylamide gel electrophoresis will be used to determine the extent of lysis or aggregation of protein samples and HPLC will be used to determine the effects of photooxidation by monitoring change in peptide maps of proteins digested with trypsin. Enzymes will also be monitored for changes in activity using assays from the literature.

许多研究人员提出，紫外线辐射可能是一种 白内障形成的致病因素。 实验模型 涉及眼部的结构蛋白（晶状体蛋白）和酶 各种动物的晶状体已被成功地用于研究 近 UV-A 辐射对体内和体外的影响。 然而，仅 最近，300-33-范围内的 UV-B 辐射受到关注。 纳米。 很少有工作将紫外线引起的变化联系起来 晶状体蛋白质和酶发生变化，例如 Na+ 和 Ca2+ 晶状体增加 在某些类型的白内障中发现的浓度。 主要 因此，这项研究的目的是观察 近UN-B引起的各种眼晶状体蛋白和酶的结构 辐射加上 UV-A 辐射，以及离子强度的变化。 这些 效果将与结构蛋白的变化相关 以及白内障形成过程中眼晶状体的酶。 为了完成这些研究，晶状体蛋白或 含有各种电解质（NaCl、KCI、CaCl2、MgSO4）的酶将 暴露于 295 nm、310 nm 或两种波长的组合 来自 450 瓦氙灯或来自脉冲氮激光器的 337.1 nm 与未经辐照的对照样品相比。 整个大鼠或牛晶状体 也会暴露在紫外线辐射下，然后酶成分会 进行隔离和分析。 色氨酸残基的光解，光产物 形成和配置的改变将使用 荧光、吸光度和傅立叶变换光谱技术 红外光谱。 从先前稳态获得的信息 荧光研究将扩展到包括寿命和各向异性 数据。 将采用SDS-聚丙烯酰胺凝胶电泳技术 确定蛋白质样品和 HPLC 的裂解或聚集程度 将用于通过监测来确定光氧化的影响 用胰蛋白酶消化的蛋白质的肽图发生变化。 酶会 还可以使用文献中的测定来监测活性的变化。