Journal
TALANTA
Volume 77, Issue 4, Pages 1497-1503Publisher
ELSEVIER
DOI: 10.1016/j.talanta.2008.09.032
Keywords
Inorganic pyrophosphate; ATP; Firefly luciferase; Ion-pair-HPLC; Bioluminescence; Experimental factorial design
Categories
Funding
- Sciences of University of Porto
- University of Porto and Caixa Geral de Depositos [IPG136]
- Fundacao para a Ciencia e Tecnologia (Lisbon) [PTDC/QUI/71366/2006]
- Fundação para a Ciência e a Tecnologia [PTDC/QUI/71366/2006] Funding Source: FCT
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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 (260nm) ion-pair-HPLC (IP-HPLC) (method A) or luciferase-dependent bioluminescence in the presence Of its Substrate, firefly luciferin (D-LH2) (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 mu M) and luciferase ([Luc] = 0.1 mu M); method B-concentrations of L-AMP ([L-AMP] = 2 mu M), luciferase ([Luc] = 50nM) and luciferin ([LH2] = 30 mu M). Method Alias a linear response over the range of 0.1-20 mu M of PPi, with a limit of detection (LOD) of 0.5 mu M and a limit of quantitation (LOQ) of 1.8 mu M. Precision, expressed as relative standard deviation (R.S.D.), is 7.4% at 1 mu M PPi and 5.9% at 8 mu M PPi. Method B has a linear response over the range of 0.75-6.0 mu M of PPi, with LOD and LOQ of 0.624 and 2.23 mu M, respectively, and a R.S.D. of 5.1% at 2.5 mu M PPi and 4.9% at 5 mu M PPi. Under optimized conditions sensitive and robust methods can be obtained for the analysis of PPi impurities in commercial nucleotides and tripolyphosphate (P-3). (C) 2008 Elsevier B.V. All rights reserved.
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