Janovská Marika, Kubala Martin, Simánek Vilím, Ulrichová Jitka
Laboratory of Biophysics, Faculty of Sciences, Palacky University, Svobody 26, 77146 Olomouc, Czech Republic.
Anal Bioanal Chem. 2009 Sep;395(1):235-40. doi: 10.1007/s00216-009-2903-9. Epub 2009 Jul 22.
The quaternary isoquinoline alkaloid, sanguinarine (SG) plays an important role in both traditional and modern medicine, exhibiting a wide range of biological activities. Under physiological conditions, there is an equilibrium between the quaternary cation (SG+) and a pseudobase (SGOH) forms of SG. In the gastrointestinal tract, SG is converted to dihydrosanguinarine (DHSG). All forms exhibit bright fluorescence. However, their spectra overlap, which limited the use of powerful techniques based on fluorescence spectroscopy/microscopy. Our experiments using a combination of steady-state and time-resolved techniques enabled the separation of individual components. The results revealed that (a) the equilibrium constant between SG+ and SGOH is pKa = 8.06, while fluorescence of DHSG exhibited no changes in the pH range 5-12, (b) the SGOH has excitation/emission spectra with maxima at 327/418 nm and excited-state lifetime 3.2 ns, the spectra of the SG+ have maxima at 475/590 nm and excited-state lifetime 2.4 ns. The DHSG spectra have maxima at 327/446 nm and 2-exponential decay with components 4.2 and 2.0 ns, (c) NADH is able to convert SG to DHSG, while there is no apparent interaction between NADH and DHSG. These techniques are applicable for monitoring the SG to DHSG conversion in hepatocytes.
季铵异喹啉生物碱血根碱（SG）在传统医学和现代医学中都发挥着重要作用，具有广泛的生物活性。在生理条件下，SG的季铵阳离子（SG+）和假碱（SGOH）形式之间存在平衡。在胃肠道中，SG会转化为二氢血根碱（DHSG）。所有形式都表现出明亮的荧光。然而，它们的光谱相互重叠，这限制了基于荧光光谱/显微镜的强大技术的应用。我们使用稳态和时间分辨技术相结合的实验实现了各个组分的分离。结果表明：（a）SG+和SGOH之间的平衡常数为pKa = 8.06，而DHSG的荧光在pH范围5 - 12内没有变化；（b）SGOH的激发/发射光谱最大值在327/418 nm，激发态寿命为3.2 ns，SG+的光谱最大值在475/590 nm，激发态寿命为2.4 ns。DHSG的光谱最大值在327/446 nm，具有4.2和2.0 ns组分的双指数衰减；（c）NADH能够将SG转化为DHSG，而NADH与DHSG之间没有明显的相互作用。这些技术适用于监测肝细胞中SG向DHSG的转化。
