Fluorescein-5-thiosemicarbazide (FTSC) labeling for fluorescent imaging of pectin-derived oligogalacturonic acid transported in living cells by confocal microscopy

Confocal microscopy is a powerful and effective tool for in vivo elucidation of the molecular basis of biological molecules. Chemical labeling of non-ultraviolet or non-fluorescent carbohydrate with fluorescent tag is an essential step that makes intra-cellular microscopic inspection possible. Howev...

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Bibliographic Details
Published in:European food research & technology 2014-11, Vol.239 (5), p.867-875
Main Authors: Zhang, Ying, Wang, Chengjian, Liu, Yang, Yao, Weinan, Sun, Yujiao, Zhang, Ping, Huang, Linjuan, Wang, Zhongfu
Format: Article
Language:English
Subjects:
Acids
Agriculture
Analytical Chemistry
Biotechnology
Byproducts
Carbohydrates
Cellular biology
Chemistry
Chemistry and Materials Science
Cytotoxicity
Fluorescence
fluorescent labeling
Food
Food Science
Forestry
human cell lines
image analysis
Labeling
Laboratories
Microscopy
Original Paper
Pectin
rats
Saccharides
Studies
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Summary:Confocal microscopy is a powerful and effective tool for in vivo elucidation of the molecular basis of biological molecules. Chemical labeling of non-ultraviolet or non-fluorescent carbohydrate with fluorescent tag is an essential step that makes intra-cellular microscopic inspection possible. However, current labeling methods have proved unsuitable for functional pectin-derived oligogalacturonic acid due to the low fluorescence intensity and/or the formation of lactone byproducts. Here, fluorescein-5-thiosemicarbazide (FTSC) was employed for fluorescent labeling of pectin-derived oligogalacturonic acid under mild and neutral reaction conditions to avoid the generation of lactone byproducts. ESI–MS and LC–MS data displayed that pectin-derived pentagalacturonic acid, GalA₅, could be quantitatively labeled with FTSC to afford thiosemicarbazones, without any lactone byproducts yielded. Moreover, GalA₅-FTSC thiosemicarbazone showed good stability and desirable fluorescence characteristics and exhibited rather low levels of cytotoxicity against sensitive rat thymus cells. Additionally, we have achieved successful fluorescent imaging of GalA₅-FTSC thiosemicarbazones transported in living SW-620 and HepG2 cells. The assay illustrates methodological significance in structure–activity elucidation study of functional carbohydrates especially acid oligo-/polysaccharides.
ISSN:1438-2377
1438-2385
DOI:10.1007/s00217-014-2283-z