PUBLICATION
Fluorescence-based detection of thiols in vitro and in vivo using dithiol probes
- Authors
- Pullela, P.K., Chiku, T., Carvan, M.J. 3rd, and Sem, D.S.
- ID
- ZDB-PUB-060315-16
- Date
- 2006
- Source
- Analytical biochemistry 352(2): 265-273 (Journal)
- Registered Authors
- Carvan III, Michael J.
- Keywords
- Thiol, Fluorescence assay, Disulfide, Escherichia coli, Zebrafish
- MeSH Terms
-
- Animals
- Cell Proliferation/drug effects
- Disulfides/chemical synthesis
- Disulfides/chemistry*
- Disulfides/pharmacokinetics
- Escherichia coli/drug effects
- Fluorescein/chemistry
- Fluorescence
- Fluorescent Dyes/chemical synthesis
- Fluorescent Dyes/chemistry*
- Fluorescent Dyes/pharmacokinetics
- Hydrogen-Ion Concentration
- In Vitro Techniques
- Molecular Structure
- Rhodamines/chemistry
- Sulfhydryl Compounds/analysis*
- Sulfhydryl Compounds/chemistry*
- Time Factors
- Tissue Distribution
- Zebrafish/embryology
- Zebrafish/growth & development
- PubMed
- 16527239 Full text @ Anal. Biochem.
Citation
Pullela, P.K., Chiku, T., Carvan, M.J. 3rd, and Sem, D.S. (2006) Fluorescence-based detection of thiols in vitro and in vivo using dithiol probes. Analytical biochemistry. 352(2):265-273.
Abstract
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 (-0.60V), it will not stress cells oxidatively. For example, 5muM 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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping