Document Detail


Conjugated polyelectrolyte-based real-time fluorescence assay for alkaline phosphatase with pyrophosphate as substrate.
MedLine Citation:
PMID:  18855416     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The fluorescence of the anionic, carboxylate-substituted poly(phenylene ethynylene) polymer PPECO2 is quenched very efficiently via the addition of 1 equiv of Cu(2+). Addition of pyrophosphate (PPi) into the weakly fluorescent solution of PPECO2 and Cu(2+) induces recovery of the polymer's fluorescence; the recovery occurs because PPi complexes with Cu(2+), effectively sequestering the ion so it cannot bind to the carboxylate groups of the polymer. A calibration curve was developed that relates the extent of fluorescence recovery to [PPi], making the PPECO2-Cu(2+) system a sensitive and selective turn-on sensor for PPi. Using the PPECO2-Cu(2+) system as the signal transducer, a real-time fluorescence turn-off assay for the enzyme alkaline phosphatase (ALP) using PPi as the substrate is developed. The assay operates with [PPi] in the micromolar range, and it offers a straightforward and rapid detection of ALP activity with the enzyme present in the nanomolar concentration range, operating either in an end point or real-time format. Kinetic and product inhibition parameters are derived by converting time-dependent fluorescence intensity into PPi (substrate) concentration, thus allowing calculation of the initial reaction rates (v(o)). Weak, nonspecific fluorescence responses are observed concomitant to addition of other proteins to the assay solution; however, the signal response to ALP is demonstrated to arise from the ALP catalyzed hydrolysis of PPi to phosphate (Pi).
Authors:
Yan Liu; Kirk S Schanze
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2008-10-15
Journal Detail:
Title:  Analytical chemistry     Volume:  80     ISSN:  1520-6882     ISO Abbreviation:  Anal. Chem.     Publication Date:  2008 Nov 
Date Detail:
Created Date:  2008-11-14     Completed Date:  2009-02-02     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370536     Medline TA:  Anal Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  8605-12     Citation Subset:  IM    
Affiliation:
Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, Florida 32611-7200, USA.
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MeSH Terms
Descriptor/Qualifier:
Alkaline Phosphatase / analysis,  antagonists & inhibitors,  metabolism*
Animals
Biocatalysis
Calibration
Cattle
Copper / chemistry
Electrolytes / chemistry*
Fluorescence*
Hydrogen-Ion Concentration
Hydrolysis
Kinetics
Phosphates / metabolism*
Polymers / chemistry*
Sensitivity and Specificity
Time Factors
Chemical
Reg. No./Substance:
0/Electrolytes; 0/Phosphates; 0/Polymers; 0/poly(phenylene ethynylene); 7440-50-8/Copper; EC 3.1.3.1/Alkaline Phosphatase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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