Dreyfuss G, Schwartz K, Blout E R, Barrio J R, Liu F T, Leonard N J
Proc Natl Acad Sci U S A. 1978 Mar;75(3):1199-203. doi: 10.1073/pnas.75.3.1199.
An approach to the study of protein receptor sites in protein mixtures or supramolecular assemblies by using fluorescence spectroscopy is described. This approach, fluorescent photoaffinity labeling, combines the merits of photoaffinity labeling to attain site-directed reactivity with the probing power of fluorescent ligands. A fluorescent photoaffinity label for cyclic AMP receptor sites of cyclic AMP-dependent protein kinases was synthesized in both unlabeled and radioactive forms. The probe, 8-azido-1,N(6)-ethenoadenosine 3',5'-cyclic monophosphate, mimics cyclic AMP in its ability to stimulate the phosphotransferase activity of the protein kinases and strongly competes with cyclic AMP for its binding sites in all preparations so far tested. Photolysis, after equilibration of protein kinase and 8-azido-1,N(6)-ethenoadenosine 3',5'-cyclic monophosphate in the dark, effects binding of the intermediate nitrene irreversibly and specifically to the cyclic AMP sites with the development of fluorescence. Excess reagent and low molecular weight photolytic products are removable by dialysis. Studies of a crude beef heart preparation containing cyclic AMP-dependent protein kinase suggest that the cyclic AMP binding sites are hydrophobic in nature and strongly immobilize the adenine moiety of the cyclic nucleotide.
本文描述了一种利用荧光光谱研究蛋白质混合物或超分子组装体中蛋白质受体位点的方法。这种方法，即荧光光亲和标记，结合了光亲和标记的优点，以实现位点特异性反应性，并具有荧光配体的探测能力。合成了一种用于环磷酸腺苷依赖性蛋白激酶环磷酸腺苷受体位点的荧光光亲和标记物，有未标记和放射性两种形式。该探针，8-叠氮基-1,N(6)-乙烯基腺苷 3',5'-环一磷酸，在刺激蛋白激酶的磷酸转移酶活性方面模拟环磷酸腺苷，并且在迄今为止测试的所有制剂中，它与环磷酸腺苷在其结合位点上有很强的竞争能力。在黑暗中使蛋白激酶与8-叠氮基-1,N(6)-乙烯基腺苷 3',5'-环一磷酸平衡后进行光解，中间态氮烯会不可逆且特异性地与环磷酸腺苷位点结合，并伴随荧光的产生。过量的试剂和低分子量光解产物可通过透析去除。对含有环磷酸腺苷依赖性蛋白激酶的粗制牛肉心制剂的研究表明，环磷酸腺苷结合位点本质上是疏水的，并且能强烈固定环核苷酸的腺嘌呤部分。
