Optimized chromatographic and bioluminescent methods for inorganic pyrophosphate based on its conversion to ATP by firefly luciferase

• Tipo: Artigo em Revista Científica Internacional
• Data: 2009

Autores

Simone M Marques (Autor) Outra; Filipe Peralta (Autor) Outra; Joaquim C G E Esteves da Silva (Autor) FCUP

Revista

Título: Talanta

Vol. 77 Nº 2

Páginas: 1497-1503

ISSN: 0039-9140

Editora: Elsevier

Indexação

ISI Web of Knowledge - 36 Citações

Scopus - 35 Citações

Classificação Científica

FOS: Ciências exactas e naturais > Química

Outras Informações

ID Authenticus: P-003-N6G

DOI: 10.1016/j.talanta.2008.09.032

Abstract (EN): 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-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 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 ([LH(2)] = 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)).

Idioma: Inglês

Tipo (Avaliação Docente): Científica

Nº de páginas: 7