Requimte, Faculdade de Ciencias, Universidade do Porto, Rua do Campo Alegre 687, 4169-007 Porto, Portugal.
Phys Chem Chem Phys. 2011 Jan 28;13(4):1521-30. doi: 10.1039/c0cp00969e. Epub 2010 Dec 10.
Outer membrane channels in gram-negative bacteria are implicated in the influx of the latest generation of cephalosporins. We have measured the interaction strengths of ceftriaxone, cefpirome and ceftazidime in the two most abundant outer membrane porins of Escherichia coli, OmpF and OmpC, by both ion current fluctuations through single protein channels and fluorescence quenching. Statistical analysis of individual antibiotic entry events in membrane-incorporated porins yielded the kinetic rates and the equilibrium binding constant of each antibiotic-porin pair. Affinity constants were independently obtained by measuring the static quenching of inherent tryptophan fluorescence in the porins in the presence of the antibiotics. Through an empirical inner filter effect correction we have succeeded in measuring the chemical interaction of these strongly absorbing antibiotics, and obtained a qualitative agreement with conductance measurements. The interaction of all three antibiotics is smaller for OmpC than OmpF, and in the case of each porin the interaction strength series ceftriaxone > cefpirome > ceftazidime is maintained.
革兰氏阴性菌的外膜通道与最新一代头孢菌素的内流有关。我们通过单个蛋白质通道的离子电流波动和荧光猝灭测量了头孢曲松、头孢匹罗和头孢他啶在大肠杆菌中最丰富的两种外膜孔蛋白 OmpF 和 OmpC 中的相互作用强度。通过对膜结合孔蛋白中单个抗生素进入事件的统计分析，得到了每个抗生素-孔蛋白对的动力学速率和平衡结合常数。通过测量抗生素存在下孔蛋白中固有色氨酸荧光的静态猝灭，独立获得了亲和力常数。通过经验性的内滤效应校正，我们成功地测量了这些强吸收抗生素的化学相互作用，并与电导测量结果一致。所有三种抗生素与 OmpC 的相互作用都小于 OmpF，对于每种孔蛋白，相互作用强度系列头孢曲松>头孢匹罗>头孢他啶保持不变。
