Khaibrakhmanova Diliara, Nikiforova Alena, Li Ziying, Sedov Igor
Chemical Institute, Kazan Federal University, Kremlevskaya 18, 420008 Kazan, Russia.
Chemical Institute, Kazan Federal University, Kremlevskaya 18, 420008 Kazan, Russia.
Int J Biol Macromol. 2022 Apr 15;204:709-717. doi: 10.1016/j.ijbiomac.2022.01.189. Epub 2022 Feb 5.
The effect of binding of several ligands to bovine serum albumin on the kinetics of fibril formation at denaturing conditions is studied. The considered ligands are clinical drugs with different binding constants to albumin: relatively strong binders (naproxen, ibuprofen, warfarin with 10 to 10 binding constant values) and weak binders (isoniazid, ranitidine with 10 to 10 binding constant values). The data of thioflavin fluorescence binding assay, Congo red binding assay, and circular dichroism spectroscopy indicate ligand concentration-dependent suppression of fibril formation in the presence of strong binders and no effects in the presence of weak binders. Analysis of kinetic curves shows no induction lag associated with fibril nucleation and the first-order kinetics of fibril formation with respect to albumin concentration for all the studied systems. Using DSC method, the fractions of unfolded albumin at incubation temperature were determined for each albumin-ligand system and ligand concentration. Their magnitudes ranging from 0 to 1 correlate with the initial rates of fibril formation and with equilibrium concentrations of fibrils formed in the system after incubation for at least 120 min. The results indicate that fibrils are formed from partially or completely denatured albumin form with the rate proportional to the fraction of this form. Strong albumin binders act as thermodynamic inhibitors of fibrillation shifting the unfolding equilibrium to the side of the native ligand-bound protein.
研究了几种配体与牛血清白蛋白结合对变性条件下纤维形成动力学的影响。所考虑的配体是与白蛋白具有不同结合常数的临床药物：相对强结合剂（萘普生、布洛芬、华法林，结合常数为10至10）和弱结合剂（异烟肼、雷尼替丁，结合常数为10至10）。硫黄素荧光结合测定、刚果红结合测定和圆二色光谱的数据表明，在强结合剂存在下，纤维形成受到配体浓度依赖性抑制，而在弱结合剂存在下则无影响。动力学曲线分析表明，所有研究系统中均不存在与纤维成核相关的诱导滞后，且纤维形成相对于白蛋白浓度呈一级动力学。使用差示扫描量热法（DSC），测定了每个白蛋白 - 配体系统和配体浓度在孵育温度下未折叠白蛋白的分数。其大小范围从0到1，与纤维形成的初始速率以及孵育至少120分钟后系统中形成的纤维的平衡浓度相关。结果表明，纤维由部分或完全变性的白蛋白形式形成，其速率与该形式的分数成正比。强白蛋白结合剂作为纤维形成的热力学抑制剂，将解折叠平衡向天然配体结合蛋白一侧移动。
