Department of Electricity, Solid Physics and Biophysics, Faculty of Physics, University of Bucharest, Măgurele, Romania, Postal address: Str. Atomistilor no. 405, CP MG - 11, Bucuresti-Magurele, RO 077125, Romania.
Department of Fundamental, Prophylactic and Clinical Disciplines, Faculty of Medicine, Transilvania University of Brasov, Brasov, Romania, Colina Universitatii no. 1, Building C, room CI30, 500068, Brasov, Romania.
Spectrochim Acta A Mol Biomol Spectrosc. 2020 Apr 5;230:118074. doi: 10.1016/j.saa.2020.118074. Epub 2020 Jan 15.
Folic acid is a bioactive food component whose deficiency can lead to a variety of health problems, while a high intake of folic acid can reduce the cytotoxicity of natural killer cells. The binding mechanism of folic acid to free bovine serum albumin (BSA) was studied using fluorescence, while the biomolecular interaction between confined-BSA and free folic acid was assessed by electrochemical methods and surface plasmon resonance. The fluorescence quenching mechanism of BSA by folic acid was found to have a static character. The thermodynamic parameters of the interaction were determined and indicated a spontaneous exothermic process with a binding constant of 8.72 × 10 M at 25 °C. Confinement of BSA to gold surfaces occurred through different immobilization methods (static and hydrodynamic), inducing conformational changes, which influenced the orientation of BSA molecules binding sites towards free folic acid. The apparent binding constant using electrochemical methods (voltammetry and impedance spectroscopy) was only 5 times higher (41 and 37 × 10 M) compared to BSA free in solution, while for surface plasmon resonance, where the hydrodynamic immobilization method was used, the value was much higher (19 × 10 M). This work gives also an insight on the interaction of BSA with gold substrates, surface plasmon resonance enabling the calculation of the adsorbed amount. The obtained results help understanding the specific interaction between free and confined BSA with free folic acid.
叶酸是一种具有生物活性的食物成分，其缺乏会导致多种健康问题，而高剂量的叶酸摄入可以降低自然杀伤细胞的细胞毒性。本研究采用荧光法研究了叶酸与游离牛血清白蛋白（BSA）的结合机制，同时采用电化学方法和表面等离子体共振评估了受限 BSA 与游离叶酸之间的生物分子相互作用。结果表明，叶酸对 BSA 的荧光猝灭机制具有静态特征。确定了相互作用的热力学参数，表明在 25°C 时是一个自发的放热过程，结合常数为 8.72×10 M。通过不同的固定化方法（静态和动态）将 BSA 限制在金表面上，会引起构象变化，从而影响 BSA 分子结合位点对游离叶酸的取向。与游离 BSA 相比，电化学方法（伏安法和阻抗谱法）的表观结合常数仅高出 5 倍（41 和 37×10 M），而对于表面等离子体共振，使用了动态固定化方法，其值则高出很多（19×10 M）。这项工作还深入了解了 BSA 与金基底的相互作用，表面等离子体共振能够计算出吸附量。所得结果有助于理解游离和受限 BSA 与游离叶酸之间的特异性相互作用。
