用长寿命金属配体络合物研究高分子量蛋白质的流体动力学

Jung Sook Kang , Grzegorz Piszczek , Joseph R. Lakowicz
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引用次数: 8

摘要

[Ru(2,2 ' -联吡啶)2(4,4 ' -二羰基-2,2 ' -联吡啶)]2+ (RuBDc)是一种非常光稳定的探针,具有良好的光物理性质,包括长寿命、高量子产率、大斯托克斯位移和高极化发射。在目前的研究中,我们证明了这种探针在监测高分子量(MW)蛋白质的旋转扩散方面的实用性。利用高强度蓝光发光二极管(LED)作为调制光源的频域荧光法,我们比较了RuBDc与免疫球蛋白G (IgG)和免疫球蛋白M (IgM)偶联的强度和各向异性衰减,结果显示其MW值相差6倍。我们获得了IgM (<τ>=428 ns缓冲液)比IgG (<τ>=422 ns缓冲液)在不存在甘油和不存在甘油时的寿命稍长。这表明在IgM中RuBDc对水的屏蔽作用比在IgG中更有效。各向异性衰减数据显示,IgM的旋转相关时间(缓冲液中为1623和65.7 ns)比IgG(缓冲液中为264和42.5 ns)更长。重要的是,缓冲液中IgM与IgG的长旋转相关次数之比为6.2,这与IgM与IgG的分子量之比(6.0)非常接近。较短的相关时间很可能与蛋白质内部的结构域运动有关。各向异性衰减反映了免疫球蛋白的分子大小和形状,以及粘度。这些结果表明RuBDc在高分子量蛋白质流体动力学研究和高分子量分析物的荧光偏振免疫分析(FPI)中具有广泛的应用。
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High-molecular-weight protein hydrodynamics studied with a long-lifetime metal-ligand complex

[Ru(2,2′-bipyridine)2(4,4′-dicarboxy-2,2′-bipyridine)]2+ (RuBDc) is a very photostable probe that possesses favorable photophysical properties including long lifetime, high quantum yield, large Stokes' shift, and highly polarized emission. In the present study, we demonstrated the usefulness of this probe for monitoring the rotational diffusion of high-molecular-weight (MW) proteins. Using frequency-domain fluorometry with a high-intensity, blue light-emitting diode (LED) as the modulated light source, we compared the intensity and anisotropy decays of RuBDc conjugated to immunoglobulin G (IgG) and immunoglobulin M (IgM), which show a six-fold difference in MW We obtained slightly longer lifetimes for IgM (<τ>=428 ns in buffer) than IgG (<τ>=422 ns in buffer) in the absence and presence of glycerol, suggesting somewhat more efficient shielding of RuBDc from water in IgM than in IgG. The anisotropy decay data showed longer rotational correlation times for IgM (1623 and 65.7 ns in buffer) as compared to IgG (264 and 42.5 ns in buffer). Importantly, the ratio of the long rotational correlation times of IgM to IgG in buffer was 6.2, which is very close to that of MW of IgM to IgG (6.0). The shorter correlation times are most likely to be associated with domain motions within the proteins. The anisotropy decays reflect both the molecular size and shape of the immunoglobulins, as well as the viscosity. These results show that RuBDc can have numerous applications in studies of high-MW protein hydrodynamics and in fluorescence polarization immunoassays (FPI) of high-MW analytes.

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