Dept. of Chemistry and Analytical Sciences, The Open University, UK; Institute of Mathematics and Statistics, Rio de Janeiro State University, Brazil.
J Photochem Photobiol B. 2013 Oct 5;127:68-77. doi: 10.1016/j.jphotobiol.2013.06.015. Epub 2013 Aug 3.
Aptamers are short, single stranded oligonucleotide or peptide molecules that bind a specific target molecule and can be used for the delivery of therapeutic agents and/or for imaging and clinical diagnosis. Several works have been developed aiming at the production of aptamers and the study of their applications, but few results have been reported on plasmatic dynamics of such products. Aptamers against the heparanase enzyme have been previously described. In this work, the interactions of two constructs of the most promising anti-heparanase aptamer (molecular weights about 9200Da and 22000Da) to human and bovine serum albumins were studied by fluorescence quenching technique. Stern-Volmer graphs were plotted and quenching constants were estimated. Stern-Volmer plots obtained from experiments carried out at 25°C and 37°C showed that the quenching of fluorescence of HSA and BSA by the low molecular weight aptamer was a collisional phenomenon (estimated Stern-Volmer constant: 3.22 (±0.01)×10(5)M(-1) for HSA at 37°C and 2.47 (±0.01)×10(5)M(-1) for HSA at 25°C), while the high molecular weight aptamer quenched albumins by static process (estimated Stern-Volmer constant: 4.05 (±0.01)×10(5)M(-1) for HSA at 37°C and 6.20 (±0.01)×10(5)M(-1) for HSA at 25°C), interacting with those proteins constituting complexes. Linear Stern-Volmer plot from HSA titrated with the low MW aptamer suggested the existence of a single binding site for the quencher in this albumin. Differently, for aptamer 2, the slightly downward curvature of the Stern-Volmer plot of the titration for that albumin suggested a possible conformational change that led to the exposition of lower affinity binding sites in HSA at 25°C. Similarly, although short aptamerdoes not appear to form a stable complex (collisional interaction), the longer aptamer is found to form a stable complex with HSA. In addition, the behaviour of quenching curves for HSA and BSA and values estimated for ratio R1/R2 from model developed by Silva et al. suggest that the primary binding site in both aptamers is located closer to the tryptophan residue in sub domain IIA. It is likely that both aptamers are competing for the same primary site in albumin.
适配体是短的、单链的寡核苷酸或肽分子，能与特定的靶分子结合，可用于治疗剂的递送和/或成像和临床诊断。已经开发了几项旨在生产适配体并研究其应用的工作，但很少有关于此类产品在血浆动力学方面的结果报道。先前已经描述了针对肝素酶的适配体。在这项工作中，通过荧光猝灭技术研究了两种最有前途的抗肝素酶适配体（分子量约为 9200Da 和 22000Da）构建体与人血清白蛋白和牛血清白蛋白的相互作用。绘制了 Stern-Volmer 图，并估算了猝灭常数。在 25°C 和 37°C 下进行实验得到的 Stern-Volmer 图表明，低分子量适配体猝灭 HSA 和 BSA 的荧光是一种碰撞现象（在 37°C 下估计的 Stern-Volmer 常数：3.22（±0.01）×10(5)M(-1)，在 25°C 下为 2.47（±0.01）×10(5)M(-1)），而高分子量适配体通过静态过程猝灭白蛋白（在 37°C 下估计的 Stern-Volmer 常数：4.05（±0.01）×10(5)M(-1)，在 25°C 下为 6.20（±0.01）×10(5)M(-1)），与构成复合物的那些蛋白质相互作用。用低 MW 适配体滴定 HSA 的线性 Stern-Volmer 图表明，在该白蛋白中存在一个用于猝灭剂的单一结合位点。不同的是，对于适配体 2，在该白蛋白的滴定的 Stern-Volmer 图中稍微向下的曲率表明可能发生了构象变化，导致在 25°C 时 HSA 中暴露了较低亲和力的结合位点。同样，尽管短适配体似乎不形成稳定的复合物（碰撞相互作用），但较长的适配体被发现与 HSA 形成稳定的复合物。此外，Silva 等人提出的模型的猝灭曲线行为和比值 R1/R2 估计值表明，这两种适配体的主要结合位点都更接近 IIA 亚域中的色氨酸残基。这两种适配体很可能都在与白蛋白中的同一主要位点竞争。
