The effect of pH on the glucose response of the glucose-galactose binding protein L255C labeled with Acrylodan.
The effect of pH on the glucose response of the glucose-galactose binding protein L255C labeled with Acrylodan.
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DOI：
10.1016/j.ijbiomac.2016.01.077
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发表时间：
2016-05
影响因子：
8.2
通讯作者：
Tolosa L
中科院分区：
文献类型：
--
作者：
El-Sayed MM;Brown SR;Mupparapu K;Tolosa L
葡萄糖-半乳糖结合蛋白 (GGBP) 在医疗和生物过程应用中用作光学生物传感器。本文研究了 pH 对 Acrylodan 标记的 GGBP-L255C 行为的影响，旨在寻找传感以及蛋白质制备、纯化和储存的最佳条件。在不同缓冲液和 pH 条件下测量不存在和存在葡萄糖时的 Acrylodan-GGBP 荧光响应。计算蛋白质-葡萄糖结合的解离常数（Kd）和吉布斯自由能（ΔG）。研究发现，在微酸性至中性条件下，尤其是接近 GBP 的等电点 (~ 5.0) 的条件下，结合更加有利。在 pH 3.0 时，对葡萄糖的荧光响应最小，并伴有稳态荧光光谱的蓝移。相比之下，在 pH 4.5 – 9.0 时，对葡萄糖的响应几乎为 45%，红移为 13 nm。频域寿命测量和 KI 猝灭表明，在高酸性条件下，无葡萄糖和葡萄糖结合蛋白的构象与在较高 pH 值下观察到的构象不同。
The glucose-galactose binding protein (GGBP) is used as an optical biosensor in medical and bioprocess applications. This paper investigates the effect of pH on the behavior of GGBP-L255C labeled with Acrylodan for the purpose of finding the optimum conditions for sensing purposes as well as for protein preparation, purification and storage. The Acrylodan-GGBP fluorescence response in absence and presence of glucose was measured under varying buffer and pH conditions. Dissociation constants (Kd) and Gibbs free energies (ΔG) for the protein-glucose binding were calculated. Binding was found to be energetically favored at slightly acidic to neutral conditions, specifically close to the pI of GBP (~ 5.0). Minimal fluorescence response to glucose was exhibited at pH 3.0 accompanied by a blue shift in the steady state fluorescence spectrum. In contrast, an almost 45% response to glucose was shown at pH 4.5 – 9.0 with a 13-nm red shift. Frequency domain lifetime measurements and quenching with KI suggest that at highly acidic conditions both the glucose-free and the glucose-bound protein are in a conformation distinct from those observed at higher pH values.