Thiol Specific and Mitochondria Selective Fluorogenic Benzofurazan Sulfide for Live Cell Nonprotein Thiol Imaging and Quantification in Mitochondria.
Anal Chem
; 90(13): 8170-8177, 2018 07 03.
Article
em En
| MEDLINE
| ID: mdl-29842788
ABSTRACT
Cellular thiols are divided into two major categories nonprotein thiols (NPSH) and protein thiols (PSH). Thiols are unevenly distributed inside the cell and compartmentalized in subcellular structures. Most of our knowledge on functions/dysfunctions of cellular/subcellular thiols is based on the quantification of cellular/subcellular thiols through homogenization of cellular/subcellular structures followed by a thiol quantification method. We would like to report a thiol-specific mitochondria-selective fluorogenic benzofurazan sulfide {7,7'-thiobis( N-rhodamine-benzo[c][1,2,5]oxadiazole-4-sulfonamide) (TBROS)} that can effectively image and quantify live cell NPSH in mitochondria through fluorescence intensity. Limited methods are available for imaging thiols in mitochondria in live cells especially in a quantitative manner. The thiol specificity of TBROS was demonstrated by its ability to react with thiols and inability to react with biologically relevant nucleophilic functional groups other than thiols. TBROS, with minimal fluorescence, formed strong fluorescent thiol adducts (λex = 550 nm, λem = 580 nm) when reacting with NPSH confirming its fluorogenicity. TBROS failed to react with PSH from bovine serum albumin and cell homogenate proteins. The high mitochondrial thiol selectivity of TBROS was achieved by its mitochondria targeting structure and its higher reaction rate with NPSH at mitochondrial pH. Imaging of mitochondrial NPSH in live cells was confirmed by two colocalization methods and use of a thiol-depleting reagent. TBROS effectively imaged NPSH changes in a quantitative manner in mitochondria in live cells. The reagent will be a useful tool in exploring physiological and pathological roles of mitochondrial thiols.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Compostos de Sulfidrila
/
Benzoxazóis
/
Microscopia de Fluorescência
/
Mitocôndrias
Limite:
Humans
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Estados Unidos