In fluorescence labeling, proteins are usually assayed to minimize the impact of fluorophores on their intrinsic structure and functions, however, the effect of the protein itself on the fluorescence properties of its attached fluorophore has not been studied at length. In this paper, the fluorescence properties of the ubiquitously used Rhodamine B (RhB) were investigated in the presence of whey proteins, particularly at alkaline conditions. Results reveal the existence of dye-protein interactions whether the dye is free or covalently linked to proteins. Alkali was able to destroy these interactions, while base-denaturation of proteins also occurred that complicates the dye-protein interactions. In addition, RhB was found able to quench the tryptophan (Trp) fluorescence of whey proteins, leading to the formation of nonfluorescent Trp-RhB complex. This stable Trp-RhB complex might explain the irreversible binding of RhB upon thermal-induced whey protein aggregates. This finding could suggest a cheaper alternative of labeling on proteins.

在荧光标记中，通常对蛋白质进行分析，以最大程度地减少荧光团对其固有结构和功能的影响，但是，尚未深入研究蛋白质本身对其附着的荧光团的荧光特性的影响。在本文中，研究了在乳清蛋白存在下，尤其是在碱性条件下，普遍使用的若丹明B（RhB）的荧光性质。结果揭示了染料-蛋白质相互作用的存在，无论染料是游离的还是与蛋白质共价连接的。碱能够破坏这些相互作用，而蛋白质的碱基变性也使染料-蛋白质相互作用复杂化。此外，发现RhB能够淬灭乳清蛋白的色氨酸（Trp）荧光，从而导致形成非荧光的Trp-RhB复合物。这种稳定的Trp-RhB复合物可能解释了RhB在热诱导的乳清蛋白聚集体上的不可逆结合。这一发现可能表明在蛋白质上标记的一种更便宜的选择。