Hantgan R R
Biochemistry. 1982 Apr 13;21(8):1821-9. doi: 10.1021/bi00537a018.
The conformation of fibrinogen in solution has been investigated by steady-state fluorescence polarization measurements. Factor XIIIa has been employed to enzymatically incorporate 1-6 mol of dansylcadaverine/mol of fibrinogen into a specific glutamine residue near the carboxy terminus of the gamma chain and up to two sites on the alpha chain. The fluorescence emission maximum of the labeled protein is shifted to 495 nm (from 538 nm for the fluorophore in solution) and the intensity substantially enhanced, indicating the covalently linked dansyl groups residue in a hydrophobic environment in the interior of the protein. This covalent modification does not interfere with the formation of fibrin, following thrombin activation. Steady-state fluorescence polarization measurements were carried out as a function of temperature and in high viscosity solvents. The fluorescent lifetime of dansylcadaverine-fibrinogen was determined by a phase shift technique. Analysis of the data by the Perrin-Weber treatment yields a rotational relaxation time of 160 ns, considerably faster than any realistic hydrodynamic model of fibrinogen would predict. The results are discussed in terms of segmental flexibility.
通过稳态荧光偏振测量研究了溶液中纤维蛋白原的构象。已使用因子ⅩⅢa将每摩尔纤维蛋白原1 - 6摩尔的丹磺酰尸胺酶促掺入γ链羧基末端附近的特定谷氨酰胺残基以及α链上的多达两个位点。标记蛋白的最大荧光发射波长移至495nm（溶液中荧光团的波长为538nm），且强度大幅增强，这表明共价连接的丹磺酰基团位于蛋白质内部的疏水环境中。这种共价修饰在凝血酶激活后不干扰纤维蛋白的形成。在不同温度和高粘度溶剂中进行了稳态荧光偏振测量。通过相移技术测定了丹磺酰尸胺 - 纤维蛋白原的荧光寿命。用佩兰 - 韦伯处理方法分析数据得出旋转弛豫时间为160ns，比纤维蛋白原任何实际的流体动力学模型预测的都要快得多。根据片段柔韧性对结果进行了讨论。
