Pullela Phani Kumar, Chiku Taurai, Carvan Michael J, Sem Daniel S
Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, Milwaukee, WI 53201, USA.
Anal Biochem. 2006 May 15;352(2):265-73. doi: 10.1016/j.ab.2006.01.047. Epub 2006 Feb 20.
Thiols play a central role in maintaining biological homeostasis. Their levels can change dramatically in response to oxidative stress associated with toxic insults, bacterial infection, and disease. Therefore, a reagent that can monitor thiol levels both in vitro and in vivo would be useful for assays and as a biomarker. Such a reagent should (i) be selective for thiols, (ii) be able to penetrate cell walls, and (iii) have a low reduction potential so as not to create oxidative stress in a cell. We have developed such a fluorescent reagent (DSSA) based on a dithiol linker: (i) the use of a dithiol linker makes it selective for thiols; (ii) the use of fluorophores that populate neutral states at physiological pH improves cell wall penetration; and (iii) because of the reagent's low reduction potential (-0.60 V), it will not stress cells oxidatively. For example, 5 microM of reagent is responsive to changes in glutathione levels in the physiologically relevant range of 1 to 10mM, yet this would oxidize less than 1% of cellular glutathione. In Escherichia coli, decreased thiol levels were detected in cells deficient in glutathione synthesis. In zebrafish embryos, the DSSA reagent permitted detection of unusually high thiol levels in the zebrafish chorion.
硫醇在维持生物体内稳态中起着核心作用。其水平会因与毒性损伤、细菌感染和疾病相关的氧化应激而发生显著变化。因此，一种能够在体外和体内监测硫醇水平的试剂对于检测和作为生物标志物都将是有用的。这样的试剂应具备：（i）对硫醇具有选择性；（ii）能够穿透细胞壁；（iii）具有较低的还原电位，以免在细胞内产生氧化应激。我们基于二硫醇连接体开发了这样一种荧光试剂（DSSA）：（i）使用二硫醇连接体使其对硫醇具有选择性；（ii）使用在生理pH值下处于中性状态的荧光团可改善细胞壁穿透性；（iii）由于该试剂的还原电位较低（-0.60 V），它不会对细胞造成氧化应激。例如，5微摩尔的试剂对1至10毫摩尔生理相关范围内的谷胱甘肽水平变化有响应，但这只会氧化不到1%的细胞内谷胱甘肽。在大肠杆菌中，检测到谷胱甘肽合成缺陷的细胞中硫醇水平降低。在斑马鱼胚胎中，DSSA试剂能够检测到斑马鱼卵膜中异常高的硫醇水平。
