Strategies for labeling proteins with PARACEST agents.
Strategies for labeling proteins with PARACEST agents.
复制标题
用 PARACEST 试剂标记蛋白质的策略。
DOI：
10.1016/j.bmc.2010.06.026
复制
发表时间：
2011
影响因子：
3.5
通讯作者：
Sherry,ADean
中科院分区：
文献类型：
--
作者：
Vasalatiy,Olga;Zhao,Piyu;Woods,Mark;Marconescu,Andrei;Castillo-Muzquiz,Aminta;Thorpe,Philip;Kiefer,GarryE;Sherry,ADean
用两种不同的PARACEST螯合物标记单克隆抗体（3G 4）和人血清白蛋白（HSA）上的反应性表面赖氨酸基团。每3G 4分子加入7.4至10.1个螯合物，每HSA分子加入5.6至5.9个螯合物，这取决于使用哪种缀合化学。通过ELISA测定测量的3G 4的免疫反应性高度依赖于附着的螯合物的数量：每个抗体7.4个螯合物的免疫反应性为88%，而每个抗体10.1个螯合物的免疫反应性仅为17%。在与3G 4缀合后，Eu-1的结合水寿命仅略微增加，从非缀合螯合物的53μs增加到缀合螯合物的65-77μs。螯合物Eu-2通过单个侧链基团与HSA缀合也导致结合水寿命几乎没有变化（缀合和非缀合形式均为73-75μs）。这些数据表明，水分子质子在共价连接的PARACEST试剂上的内球位点和本体水之间的交换在很大程度上不受试剂与蛋白质的连接模式以及可能的蛋白质表面上的化学环境的影响。
Reactive surface lysine groups on the monoclonal antibody (3G4) and on human serum albumin (HSA) were labeled with two different PARACEST chelates. Between 7.4 and 10.1 chelates were added per 3G4 molecule and between 5.6 and 5.9 chelates per molecule of HSA, depending upon which conjugation chemistry was used. The immunoreactivity of 3G4 as measured by ELISA assays was highly dependent upon the number of attached chelates: 88% immunoreactivity with 7.4 chelates per antibody versus only 17% immunoreactivity with 10.1 chelates per antibody. Upon conjugation to 3G4, the bound water lifetime of Eu-1 increased only marginally, up from 53μs for the non-conjugated chelate to 65–77μs for conjugated chelates. Conjugation of a chelate Eu-2 to HSA via a single side-chain group also resulted in little or no change in bound water lifetime (73–75μs for both the conjugated and non-conjugated forms). These data indicate that exchange of water molecules protons between the inner-sphere site on covalently attached PARACEST agent and bulk water is largely unaffected by the mode of attachment of the agent to the protein and likely its chemical surroundings on the surface of the protein.