Katzenellenbogen J A, Ruh T S, Carlson K E, Iwamoto H S, Gorski J
Biochemistry. 1975 Jun 3;14(11):2310-6. doi: 10.1021/bi00682a006.
Ultraviolet irradiation of the estrogen binding protein in rat uterine cytosol results in a progressive photoinactivation which is rapid at 254 nm and slower at greater than 315 nm. Both unfilled and estradiol-filled sites are inactivated at approimately the same rates at 254 nm (t 1/2 equals 8 min and 11 min, respectively), but at 315 nm, empty sites are consumed much more rapidly (t 1/2 equals 3.4 hr) than filled ones (t 1/2 equals 24 hr). The protective effect of the estrogen ligand at this wavelength appears to depend on its binding to the estrogen-specific binding site, as inactivation rate studies at different concentrations of estrone, estradiol, and estriol show a good correlation between the extent of protection and the fractional saturation of the high affinity estrogen binding sites. Scatchard analysis indicates that inactivation is the result of a loss of binding sites and not a decrease in their affinity, and sedimentation analysis shows that increased heterogeneity and aggregation of the estrogen binding species accompanies the photoinactivation process. Photoinactivation appears to be the result of direct irradiative damage of the animo acid residues, as the inactivation rate is the same under air and nitrogen atmospheres, and is unaffected by nucleophiles, reductants, and radical scavengers. When photoinactivation is measureed by irradiation of cytosol containing [3-H]estradiol, a concurrent photocovalent attachment process is noted; the steroid becomes linked to protein in a solvent-extractable manner (boiling ethanol inextractable). This attachment, however, does not appear to be related to the steroid binding at the estrogen binding site. Its rate is affected by reductants and scavengers. A similar photocovalent attachment reaction occurs when bovine serum albumin or ovalbumin is irradiated in the presence of [3-H]estradiol or [3-H]diethylstilbestrol. The detailed reactions involved in this photocovalent attachment process have not been defined at present.
对大鼠子宫胞质溶胶中的雌激素结合蛋白进行紫外线照射会导致渐进性光灭活，在254纳米处这种光灭活很快，而在大于315纳米处则较慢。在254纳米处，未占据位点和雌二醇占据位点的失活速率大致相同（半衰期分别为8分钟和11分钟），但在315纳米处，空位点的消耗速度比占据位点快得多（半衰期为3.4小时），而占据位点的半衰期为24小时。雌激素配体在该波长下的保护作用似乎取决于其与雌激素特异性结合位点的结合，因为在不同浓度的雌酮、雌二醇和雌三醇下进行的失活速率研究表明，保护程度与高亲和力雌激素结合位点的分数饱和度之间存在良好的相关性。Scatchard分析表明，失活是结合位点丧失的结果，而非其亲和力降低，沉降分析表明，雌激素结合物种的异质性增加和聚集伴随着光灭活过程。光灭活似乎是氨基酸残基直接辐射损伤的结果，因为在空气和氮气气氛下失活速率相同，并且不受亲核试剂、还原剂和自由基清除剂的影响。当通过照射含有[3-H]雌二醇的胞质溶胶来测量光灭活时，会注意到同时发生的光共价附着过程；类固醇以可被溶剂提取的方式（不可被沸腾乙醇提取）与蛋白质相连。然而，这种附着似乎与雌激素结合位点处的类固醇结合无关。其速率受还原剂和清除剂的影响。当在[3-H]雌二醇或[3-H]己烯雌酚存在下照射牛血清白蛋白或卵清蛋白时，会发生类似的光共价附着反应。目前尚未确定该光共价附着过程中涉及的详细反应。
