Jisha Vadakkancheril S, Arun Kalliat T, Hariharan Mahesh, Ramaiah Danaboyina
Photosciences and Photonics, Regional Research Laboratory (CSIR), Trivandrum 695019, India.
J Am Chem Soc. 2006 May 10;128(18):6024-5. doi: 10.1021/ja061301x.
With the objective of developing small molecule based probes for proteins, interactions of polyhydroxyl-substituted squaraine dye (SQ) with bovine serum albumin (BSA) have been investigated by absorption, steady-state and time-resolved fluorescence, circular dichroism (CD), cyclic voltammetry (CV), 1H NMR, scanning electron, and tapping mode atomic force microscopic techniques. Increase in addition of BSA resulted in increase in absorbance and fluorescence quantum yields (80-fold) of SQ, along with significant bathochromic shifts in the absorption and fluorescence maxima. Half-reciprocal analysis of the absorption data gave a 1:1 stoichiometry for the complex between BSA and SQ with high association (Kass) constant of (1.4 +/- 0.1) x 106 M-1 and change in free energy of -35 kJ/mol. The complex formation was further confirmed by observation of induced CD signal corresponding to the SQ chromophore at 610 nm, upfield shift (about Deltadelta 0.1 ppm) of aromatic protons of SQ in 1H NMR spectra, and decrease in current intensity (CV) of SQ when bound to BSA. The picosecond time-resolved fluorescence studies indicated that the BSA-SQ complex exhibits biexponential decay with significantly enhanced lifetimes of 0.5 and 1.5 ns when compared to the lifetime of SQ (tau = 121 ps) in the absence of BSA. Employing displacement cum fluorimetry using site-specific binding ligands, such as dansylproline and dansylamide, indicated that SQ binds with protein selectively at site II involving hydrophobic, hydrogen bonding, and electrostatic interactions. The uniqueness of this molecular system is that it interacts with BSA selectively at site II and signals the binding event through dual mode recognition of "visual color" change and "turn on" fluorescence mechanism.
为了开发基于小分子的蛋白质探针，通过吸收光谱、稳态和时间分辨荧光光谱、圆二色光谱（CD）、循环伏安法（CV）、1H NMR、扫描电子显微镜和敲击模式原子力显微镜技术，研究了多羟基取代方酸染料（SQ）与牛血清白蛋白（BSA）的相互作用。加入BSA后，SQ的吸光度和荧光量子产率增加（80倍），同时吸收峰和荧光峰发生显著的红移。对吸收数据进行半倒数分析得出，BSA与SQ形成的复合物化学计量比为1:1，缔合常数（Kass）高，为(1.4±0.1)×106 M-1，自由能变化为-35 kJ/mol。在610 nm处观察到对应于SQ发色团的诱导CD信号、1H NMR谱中SQ芳香质子的高场位移（约Δδ 0.1 ppm）以及与BSA结合时SQ电流强度的降低（CV），进一步证实了复合物的形成。皮秒时间分辨荧光研究表明，与无BSA时SQ的寿命（τ = 121 ps）相比，BSA-SQ复合物呈现双指数衰减，寿命显著延长至0.5和1.5 ns。使用位点特异性结合配体（如丹磺酰脯氨酸和丹磺酰胺）进行置换荧光测定表明，SQ在涉及疏水、氢键和静电相互作用的位点II选择性地与蛋白质结合。该分子系统的独特之处在于它在位点II选择性地与BSA相互作用，并通过“视觉颜色”变化和“开启”荧光机制的双重模式识别来指示结合事件。
