Sharma Jai, Tleugabulova Dina, Czardybon Wojciech, Brennan John D
Department of Chemistry, McMaster University, Hamilton, ON, L8S 4M1, Canada.
J Am Chem Soc. 2006 Apr 26;128(16):5496-505. doi: 10.1021/ja058707e.
Time-resolved fluorescence anisotropy (TRFA) is widely used to study dynamic motions of biomolecules in a variety of environments. However, depolarization due to rapid side chain motions often complicates the interpretation of anisotropy decay data and interferes with the accurate observation of segmental motions. Here, we demonstrate a new method for two-point ionic labeling of polymers and biomolecules that have appropriately spaced amino groups using the fluorescent probe 8-hydroxyl-1,3,6-trisulfonated pyrene (pyranine). TRFA analysis shows that such labeling provides a more rigid attachment of the fluorophore to the macromolecule than the covalent or single-point ionic labeling of amino groups, leading to time-resolved anisotropy decays that better reflect the backbone motion of the labeled polymer segment. Optimal coupling of pyranine to biomolecule dynamics is shown to be obtained for appropriately spaced Arg groups, and in such cases the ionic binding is stable up to 150 mM ionic strength. TRFA was used to monitor the behavior of pyranine-labeled poly(allylamine) (PAM) and poly-d-lysine (PL) in sodium silicate derived sol-gel materials and revealed significant restriction of backbone motion upon entrapment for both polymers, an observation that was not readily apparent in a previous study with entrapped fluorescein-labeled PAM and PL. The implications of these findings for fluorescence studies of polymer and biomolecule dynamics are discussed.
时间分辨荧光各向异性（TRFA）被广泛用于研究各种环境中生物分子的动态运动。然而，由于快速的侧链运动导致的去极化常常使各向异性衰减数据的解释变得复杂，并干扰对片段运动的准确观察。在此，我们展示了一种使用荧光探针8-羟基-1,3,6-三磺酸芘（吡喃荧光素）对具有适当间隔氨基的聚合物和生物分子进行两点离子标记的新方法。TRFA分析表明，与氨基的共价或单点离子标记相比，这种标记使荧光团与大分子的连接更加刚性，从而导致时间分辨各向异性衰减能更好地反映标记聚合物片段的主链运动。对于适当间隔的精氨酸基团，已证明吡喃荧光素与生物分子动力学的最佳偶联，在这种情况下，离子结合在高达150 mM的离子强度下都是稳定的。TRFA被用于监测吡喃荧光素标记的聚烯丙胺（PAM）和聚-D-赖氨酸（PL）在硅酸钠衍生的溶胶-凝胶材料中的行为，并揭示了两种聚合物在被包埋时主链运动受到显著限制，这一观察结果在先前关于被包埋的荧光素标记的PAM和PL的研究中并不明显。讨论了这些发现对聚合物和生物分子动力学荧光研究的意义。
