Sensitizer-induced conformational changes in lens crystallin--I. Photodynamic action of methylene blue and N-formylkynurenine on bovine alpha-crystallin.
Sensitizer-induced conformational changes in lens crystallin--I. Photodynamic action of methylene blue and N-formylkynurenine on bovine alpha-crystallin.
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敏化剂诱导的晶状体晶状体蛋白构象变化--I．
DOI：
10.1111/j.1751-1097.1986.tb09529.x
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发表时间：
1986
影响因子：
3.3
通讯作者：
Chakrabarti,B
中科院分区：
文献类型：
--
作者：
Mandal,K;Bose,SK;Chakrabarti,B
牛α-晶状体蛋白的荧光和圆二色谱性质已被监测，以检测在增敏剂亚甲基蓝或N-甲酰基犬尿氨酸存在下光反应后蛋白质结构完整性的变化。亚甲基蓝敏化的光氧化导致了近紫外光CD的三级结构的变化，这种变化在照射后0.5h内观察到，在这段时间内色氨酸的释放迅速减少。使用针对氧的活性物种的抑制剂，已经表明单线态氧主要引起这种变化，但敏化剂分子也在这一过程中起到一定的作用。在亚甲基蓝存在的情况下，在有氧和无氧条件下照射6h后，位于蛋白质非极性区域的硫醇基团暴露在极性环境中。与这些荧光结果一致。近紫外光Cd(三级结构)发生剧烈变化，而远紫外光Cd(二级结构)基本保持不变。对抑制剂的研究表明，增敏剂分子本身对这一过程起主要作用。构象的这种主要变化的解释是，大部分蛋白质在光敏反应中展开，从而改变了不同氨基酸的微环境、取向和分子间相互作用。N-甲酰犬尿氨酸在近紫外光CD中也表现出一些变化，可能是由于光敏化反应中产生的过氧化氢引起的。但在亚甲基蓝的情况下观察到的硫醇微环境和近紫外CD的主要变化，即使当蛋白质在N-甲酰基犬尿氨酸和空气存在下照射6h也不会发生。
Fluorescence and circular dichroic properties of bovine a‐crystallin have been monitored to detect changes in the structural integrity of the protein following photoreactions in the presence of sensitizer, either methylene blue or N‐formylkynurenine. Methylene blue‐sensitized photooxidation causes a change in the tertiary structure as manifested in the near‐UV CD; this is observed within 0.5 h of irradiation during which time tryptophan emission decreases rapidly. Using inhibitors specific for active species of oxygen, it has been shown that singlet oxygen predominantly causes this change but the sensitizer molecules also have some role in this process. Upon 6 h of irradiation in the presence of methylene blue under both aerobic and anaerobic conditions, the thiol groups that were in a non‐polar region of the protein are exposed to polar environments. In conformity with these fluorescence results. near‐UV CD (tertiary structure) suffers a drastic alteration whereas the far‐UV CD (secondary structure) remains virtually unchanged. The studies with inhibitors indicate that sensitizer molecule itself is primarily responsible for this process. This major change in the conformation has been explained by suggesting that a large portion of the protein unfolds in the photosensitized reaction, thereby altering microenviron‐ments, orientation, and intermolecular interactions of different amino acids. N‐formylkynurenine also shows some changes in the near‐UV CD, presumably, caused by H2O2generated in the photosensitized reaction. But the major alteration in the microenvironments of thiol groups and in the near‐UV CD, as observed in the case of methylene blue, does not occur even when the protein is irradiated for 6 h in the presence of N‐formylkynurenine and air.