Terpetschnig E, Szmacinski H, Malak H, Lakowicz J R
Department of Biological Chemistry, University of Maryland, Baltimore School of Medicine 21201.
Biophys J. 1995 Jan;68(1):342-50. doi: 10.1016/S0006-3495(95)80193-1.
We describe the use of asymmetric Ru-ligand complexes as a new class of luminescent probes that can be used to measure rotational motions of proteins. These complexes are known to display luminescent lifetimes ranging from 10 to 4000 ns. In this report, we show that the asymmetric complex Ru(bpy)2(dcbpy) (PF6)2 displays a high anisotropy value when excited in the long wavelength absorption band. For covalent linkage to proteins, we synthesized the N-hydroxy succinimide ester of this metal-ligand complex. To illustrate the usefulness of these probes, we describe the intensity and anisotropy decays of [Ru(bpy)2(dcbpy)] when covalently linked to human serum albumin, concanavalin A (ConA), human immunoglobulin G (IgG), and Ferritin, and measured in solutions of increased viscosity. These data demonstrate that the probes can be used to measure rotational motions on the 10 ns to 1.5 microseconds timescale, which so far has been inaccessible using luminescence methods. The present probe [Ru(bpy)2(dcbpy)] can be regarded as the first of a class of metal-ligand complexes, each with different chemical reactivity and spectral properties, for studies of macromolecular dynamics.
我们描述了使用不对称钌-配体配合物作为一类新型发光探针，可用于测量蛋白质的旋转运动。已知这些配合物的发光寿命在10到4000纳秒之间。在本报告中，我们表明不对称配合物Ru(bpy)2(dcbpy)(PF6)2在长波长吸收带激发时显示出高各向异性值。为了与蛋白质共价连接，我们合成了这种金属-配体配合物的N-羟基琥珀酰亚胺酯。为了说明这些探针的实用性，我们描述了[Ru(bpy)2(dcbpy)]与人类血清白蛋白、伴刀豆球蛋白A（ConA）、人类免疫球蛋白G（IgG）和铁蛋白共价连接并在粘度增加的溶液中测量时的强度和各向异性衰减。这些数据表明，这些探针可用于测量10纳秒到1.5微秒时间尺度上的旋转运动，而到目前为止，使用发光方法还无法实现这一点。目前的探针[Ru(bpy)2(dcbpy)]可被视为一类金属-配体配合物中的第一种，每种配合物具有不同的化学反应性和光谱特性，用于研究大分子动力学。
