Szmacinski H, Castellano F N, Terpetschnig E, Dattelbaum J D, Lakowicz J R, Meyer G J
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore 21201, USA.
Biochim Biophys Acta. 1998 Mar 3;1383(1):151-9. doi: 10.1016/s0167-4838(97)00196-9.
We describe the synthesis and characterization of two asymmetrical ruthenium(II) complexes, [Ru(dpp)2(dcbpy)]2+ and [Ru(dpp)2(mcbpy)]2+, as well as the water soluble sulfonated derivatives [Ru(dpp(SO3Na)2)2(dcbpy)]2+ and [Ru(dpp(SO3Na)2)2(mcbpy)]2+ (dpp is 4,7-diphenyl-1,10-phenanthroline, dcbpy is 4,4'-dicarboxylic acid-2,2'-bipyridine, mcbpy is 4-methyl,4'-carboxylic acid-2,2'-bipyridine, and dpp(SO3Na)2 is the disulfonated derivative of dpp) as probes for the measurement of the rotational motions of proteins. The spectral (absorption, emission, and anisotropy) and photophysical (time-resolved intensity and anisotropy decays) properties of these metal-ligand complexes were determined in solution, in both the presence and absence of human serum albumin (HSA). These complexes display lifetimes ranging from 345 ns to 3.8 microseconds in deoxygenated aqueous solutions under a variety of conditions. The carboxylic acid groups on these complexes were activated to form N-hydroxysuccinimide (NHS) esters which were used to covalently lable HSA, and were characterized spectroscopically in the same manner as above. Time-resolved anisotropy measurements were performed to demonstrate the utility of these complexes in measuring long rotational correlation times of bioconjugates between HSA and antibody to HSA. The potential usefulness of these probes in fluorescence polarization immunoassays was demonstrated by an association assay of the Ru(II)-labeled HSA with polyclonal antibody.
我们描述了两种不对称钌（II）配合物[Ru(dpp)2(dcbpy)]2+和[Ru(dpp)2(mcbpy)]2+的合成与表征，以及水溶性磺化衍生物[Ru(dpp(SO3Na)2)2(dcbpy)]2+和[Ru(dpp(SO3Na)2)2(mcbpy)]2+（dpp为4,7-二苯基-1,10-菲咯啉，dcbpy为4,4'-二羧酸-2,2'-联吡啶，mcbpy为4-甲基-4'-羧酸-2,2'-联吡啶，dpp(SO3Na)2为dpp的二磺化衍生物）作为测量蛋白质旋转运动的探针。在有无人血清白蛋白（HSA）存在的情况下，在溶液中测定了这些金属-配体配合物的光谱（吸收、发射和各向异性）和光物理性质（时间分辨强度和各向异性衰减）。在各种条件下，这些配合物在脱氧水溶液中的寿命范围为345纳秒至3.8微秒。这些配合物上的羧酸基团被活化形成N-羟基琥珀酰亚胺（NHS）酯，用于共价标记HSA，并以与上述相同的方式进行光谱表征。进行了时间分辨各向异性测量，以证明这些配合物在测量HSA与抗HSA抗体之间生物共轭物的长旋转相关时间方面的效用。通过Ru（II）标记的HSA与多克隆抗体的结合试验，证明了这些探针在荧光偏振免疫分析中的潜在用途。
