Ruoho A E, Kiefer H, Roeder P E, Singer S J
Proc Natl Acad Sci U S A. 1973 Sep;70(9):2567-71. doi: 10.1073/pnas.70.9.2567.
Photoaffinity labeling is a recently introduced method for covalently binding chemical tags to the active sites of protein molecules, which is potentially capable of very great specificities of labeling. A labeling reagent is used that is converted by photolysis to an extremely reactive intermediate. According to the expected mechanism, the reagent molecules that are specifically and reversibly bound to the active site at the instant of photolysis react irreversibly in the site before they can dissociate from the site. In two such reagent-protein systems studied in this paper, however, it is shown that, while by the usual criteria photoaffinity labeling appears to have occurred, the expected mechanism in fact does not hold. This was discovered in experiments with scavengers present in the mixtures that were photolyzed. The general properties of, and criteria for, photoaffinity labeling reactions are discussed in the light of these findings.
光亲和标记是一种最近引入的将化学标签共价结合到蛋白质分子活性位点的方法，它有可能实现非常高的标记特异性。使用一种标记试剂，该试剂通过光解转化为极具反应性的中间体。根据预期机制，在光解瞬间特异性且可逆地结合到活性位点的试剂分子，在它们能够从该位点解离之前就在该位点发生不可逆反应。然而，在本文研究的两个这样的试剂 - 蛋白质系统中，结果表明，虽然按照通常标准似乎发生了光亲和标记，但预期机制实际上并不成立。这是在对光解混合物中存在清除剂的实验中发现的。根据这些发现，讨论了光亲和标记反应的一般性质和标准。
