IJUP08 - Universidade do Porto

Optimization of an enzymatic and chromatographic method for
inorganic pyrophosphate based on firefly luciferase
Simone M. Marques 1 and Joaquim C.G. Esteves da Silva 1
1 Department of Chemistry, CIQ-UP, Faculty of Sciences, University of Porto, Portugal.
Inorganic pyrophosphate (PPi) is a compound generated mainly in anabolic pathways.
Although PPi was initially regarded as a by-product its biological importance is now well
established. In this communication a new method for quantifying PPi is described. This
method is based on the production of ATP (and L - dehydroluciferin) from PPi and
dehydroluciferyl-adenylate (L-AMP) catalyzed by firefly luciferase (EC 1.13.12.7) (Eq.1)
[1].
L-AMP + PPi ⇌ L + ATP (1)
The ATP formed can be identified and quantified by ion pair-HPLC with detection at 260
nm.
The method was subjected to an optimization using experimental design methodologies to
obtain suitable values for the identified factors: incubation time (tinc), inactivation time of
the enzyme (tinac), pH of the assay (pH) and the concentrations of L-AMP (LAMP) and
luciferase (Luc). Fig. 1 shows a response surface for the two observed critical factors:
LAMP and tinc.
Fig.1 Response surface for the critical factors.
The optimized method is linear over a range of 0.1-10 μM PPi, with a limit of detection
(LOD) of 1.2 μM and a limit of quantification (LOQ) of 3.9 μM.
Acknowledgements: Financial support from Universidade do Porto and Caixa Geral de
Depósitos (Project IPG136) is acknowledged.
References:
[1] Fraga, H., Fernandes, D., Novotny, J., Fontes, R. and Esteves da Silva, J.C.G. (2006), Firefly
Luciferase Produces Hydrogen Peroxide as a Coproduct in Dehydroluciferyl Adenylate Fornation,
ChemBioChem, 7 (6), 929-935.
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