Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; Department of Animal Radiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Mar 15;1141:122025. doi: 10.1016/j.jchromb.2020.122025. Epub 2020 Feb 25.
Cd(II) is toxic to many species, including humans, because it inactivates a number of enzymes and induces cytopathic effects in the liver, kidney, and skeletal tissues in humans. Metallothionein and glutathione (GSH) play a major role in the protection against Cd(II)-induced toxicity in mammalian cells. In this study, a relatively simple method for detecting trace amounts of Cd(II) chelators was developed by using 5,10,15,20-tetraphenyl-21H,23H-porphinetetrasulfonic acid (TPPS). The TPPS-Cd(II) complex was added to the elutions of high-performance liquid chromatography. The Cd(II) chelators separated by column chromatography were mixed with Cd(II)-bound TPPS (TPPS-Cd(II)). Cd(II) from TPPS-Cd(II) was chelated by the eluted Cd(II) chelators, resulting in the formation of free TPPS. The absorbance of TPPS shifted from 434 nm (TPPS-Cd(II)) to 414 nm (TPPS), and this characteristic shift was used to estimate the quantity and affinity of the Cd(II) chelators. This new method was compared with the bathocuproine disulfonate (BCS) method developed in our previous study. Instead of BCS-Cu(I), TPPS-Cd(II) was used as the colorimetric reagent. The experimental setup of the TPPS-based method is more general, and the preparation of the colorimetric solution is also much simpler than the BCS method. To verify the efficacy of this new method, we determined the actual Cd(II)-chelating ability of GSH in horse blood; the obtained concentration was in good agreement with the previously reported value.
Cd(II) 对许多物种具有毒性，包括人类，因为它会使许多酶失活，并在人类的肝脏、肾脏和骨骼组织中引起细胞病变效应。金属硫蛋白和谷胱甘肽 (GSH) 在哺乳动物细胞对抗 Cd(II) 诱导的毒性中起着重要作用。在这项研究中，我们开发了一种相对简单的方法，使用 5,10,15,20-四苯基-21H,23H-卟啉四磺酸（TPPS）来检测痕量 Cd(II) 螯合剂。将 TPPS-Cd(II) 复合物添加到高效液相色谱的洗脱液中。通过柱层析分离的 Cd(II) 螯合剂与结合 Cd(II) 的 TPPS（TPPS-Cd(II)）混合。从 TPPS-Cd(II) 中释放出的 Cd(II) 被洗脱的 Cd(II) 螯合剂螯合，形成游离的 TPPS。TPPS 的吸光度从 434nm（TPPS-Cd(II)）转移到 414nm（TPPS），这种特征转移被用来估计 Cd(II) 螯合剂的数量和亲和力。这种新方法与我们之前研究中开发的浴铜灵二磺酸盐（BCS）方法进行了比较。与 BCS-Cu(I) 不同，TPPS-Cd(II) 被用作比色试剂。TPPS 法的实验装置更通用，比色溶液的制备也比 BCS 法简单得多。为了验证这种新方法的有效性，我们测定了马血中 GSH 的实际 Cd(II) 螯合能力；得到的浓度与之前报道的值非常吻合。
