Optimized chromatographic and bioluminescent methods for inorganic pyrophosphate based on its conversion to ATP by firefly luciferase
| Autor: | Filipe Peralta, Simone M. Marques, Joaquim C.G. Esteves da Silva |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
Luminescence
Time Factors Firefly luciferin High-performance liquid chromatography Analytical Chemistry chemistry.chemical_compound Adenosine Triphosphate Bioluminescence Animals Luciferase Luciferases Detection limit Ions Chromatography Chemistry Nucleotides Fireflies Substrate (chemistry) Reproducibility of Results Hydrogen-Ion Concentration Models Theoretical Luciferin Diphosphates Models Chemical Calibration Quantitative analysis (chemistry) |
| Zdroj: | Talanta. 77(4) |
| ISSN: | 1873-3573 |
| Popis: | Two new methods for inorganic pyrophosphate (PPi) quantification are described. They are based on the enzymatic conversion of PPi into ATP by firefly luciferase (Luc, E.C. 1.13.12.7) in the presence of dehydroluciferyl-adenylate (L-AMP) followed by the determination of ATP by one of two different procedures, either UV-monitored (260 nm) ion-pair-HPLC (IP-HPLC) (method A) or luciferase-dependent bioluminescence in the presence of its substrate, firefly luciferin (D-LH(2)) (method B). These methods were subjected to optimization using experimental design methodologies to obtain optimum values for the selected factors: method A-incubation time (t(inc)=15 min), inactivation time of the enzyme (t(inac)=2 min), pH of the reaction mixture (pH 7.50) and the concentrations of L-AMP ([L-AMP]=40 microM) and luciferase ([Luc]=0.1 microM); method B-concentrations of L-AMP ([L-AMP]=2 microM), luciferase ([Luc]=50 nM) and luciferin ([LH(2)]=30 microM). Method A has a linear response over the range of 0.1-20 microM of PPi, with a limit of detection (LOD) of 0.5 microM and a limit of quantitation (LOQ) of 1.8 microM. Precision, expressed as relative standard deviation (R.S.D.), is 7.4% at 1 microM PPi and 5.9% at 8 microM PPi. Method B has a linear response over the range of 0.75-6.0 microM of PPi, with LOD and LOQ of 0.624 and 2.23 microM, respectively, and a R.S.D. of 5.1% at 2.5 microM PPi and 4.9% at 5 microM PPi. Under optimized conditions sensitive and robust methods can be obtained for the analysis of PPi impurities in commercial nucleotides and tripolyphosphate (P(3)). |
| Databáze: | OpenAIRE |
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