Andley U P, Clark B A
Howe Laboratory of Ophthalmology, Harvard Medical School, Boston, MA.
Curr Eye Res. 1988 Jun;7(6):571-9. doi: 10.3109/02713688809031813.
The photooxidation of calf gamma-crystallin has been investigated in (a) riboflavin-sensitized reaction and (b) direct photolysis of the tryptophan residues of the protein at 300 nm. Partial insolubilization of the protein is evident by the increase in turbidity of the photolyzed protein solution. The turbidity is diminished when anaerobic conditions are used for irradiation. Spectroscopic studies on the soluble phase of the photolyzed protein reveal significant changes in the near-UV circular dichroism spectrum, suggesting that changes in the tertiary structure of the protein precede insolubilization. Isoelectric focusing analyses reveal that the gamma-crystallin polypeptides have more acidic pI's after photooxidation under both conditions. The insoluble protein is highly crosslinked into dimers and higher oligomers via covalent, non-disulfide crosslinks, but inter-subunit crosslinking is negligible in the soluble phase of the photolyzed protein. The mechanism of photooxidation involves the generation of H2O2 in the riboflavin-sensitized reaction. In the direct photolysis of tryptophan residues by 300 nm irradiation, there is a rapid disruption of the protein structure, apparently by the oxidation of the hydrophobic tryptophan residues.
已对小牛γ-晶体蛋白的光氧化进行了研究，具体如下：(a) 核黄素敏化反应；(b) 在300 nm波长下对该蛋白质的色氨酸残基进行直接光解。光解后的蛋白质溶液浊度增加，表明蛋白质出现了部分不溶性。在厌氧条件下进行辐照时，浊度会降低。对光解后蛋白质的可溶相进行光谱研究发现，近紫外圆二色光谱有显著变化，这表明蛋白质三级结构的变化先于其不溶性的出现。等电聚焦分析表明，在两种条件下光氧化后，γ-晶体蛋白多肽的等电点（pI）更偏酸性。不溶性蛋白质通过共价、非二硫键交联高度交联成二聚体和更高的寡聚体，但在光解后蛋白质的可溶相中，亚基间交联可忽略不计。光氧化机制包括在核黄素敏化反应中生成过氧化氢。在300 nm辐照直接光解色氨酸残基时，蛋白质结构迅速遭到破坏，显然是由于疏水性色氨酸残基被氧化所致。
