Exploring albumin-sulfonephthalein dyes interactions by a chemometric-assisted and multi-technique equilibria analysis in solution.

Lisa Rita Magnaghi, Marta Guembe-Garcia, Giuseppina D G Santonoceta, Andrea Capucciati, Paolo Quadrelli, Carmelo Sgarlata, Giancarla Alberti, Raffaela Biesuz
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Abstract

Albumin is undoubtedly the most studied protein thanks to its widespread diffusion and biochemistry; despite its binding ability towards different dyes, provoking dye's colour change, has been exploited for decades for quantification purposes, the joint effect of working pH, ionic strength, and dye's pKa still remains only sporadically discussed. In the present study, the interaction of Bovine Serum Albumin (BSA) with five dyes belonging to the sulfonephthalein group, Bromophenol Blue (BPB, pKa = 3.75), Bromocresol Green (BCG, pKa = 4.42), Chlorophenol Red (CPR, pKa = 5.74), Bromocresol Purple (BCP, pKa = 6.05) and Bromothymol Blue (BTB, pKa = 6.72), is investigated at four working pH values (3.5, 6.0, 7.5 and 9.0) and two ionic strength conditions by UV-Vis spectroscopy. Principal Component Analysis is then applied to rationalize dye behavior upon BSA addition at each pH value and to summarize the protein effect on dyes' spectral features, identifying three general behaviors. The most relevant systems are then submitted to further characterization involving a solution equilibria study aimed at determining conditional binding constants for the selected DSA-dye adducts and fluorescence, CD, and 1H NMR spectroscopy to evaluate the binding effect on the species involved.

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通过溶液中的化学计量辅助和多技术平衡分析,探索白蛋白-磺酞染料之间的相互作用。
由于白蛋白具有广泛的扩散性和生物化学性,它无疑是研究最多的蛋白质;尽管白蛋白与不同染料的结合能力(引起染料颜色变化)已被用于定量分析目的达数十年之久,但有关工作 pH 值、离子强度和染料 pKa 的共同作用的讨论仍然十分零散。在本研究中,牛血清白蛋白(BSA)与属于磺酞类的五种染料--溴酚蓝(BPB,pKa = 3.75)、溴甲酚绿(BCG,pKa = 4.42)、氯酚红(CPR,pKa = 5.74)、溴甲酚紫(BCP,pKa = 6.05)和溴百里酚蓝(BTB,pKa = 6.72)。然后,应用主成分分析法对每个 pH 值下添加 BSA 后的染料行为进行合理分析,并总结蛋白质对染料光谱特征的影响,从而确定三种一般行为。然后对最相关的系统进行进一步表征,包括溶液平衡研究,旨在确定所选 DSA-染料加合物的条件结合常数,以及荧光、CD 和 1H NMR 光谱,以评估对相关物种的结合效应。
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