Marques Simone M, Peralta Filipe, Esteves da Silva Joaquim C G
Department of Chemistry, Faculty of Sciences, Centro de Investigação em Química, University of Porto, Porto, Portugal.
Talanta. 2009 Feb 15;77(4):1497-503. doi: 10.1016/j.talanta.2008.09.032. Epub 2008 Sep 27.
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)).
本文描述了两种用于无机焦磷酸（PPi）定量的新方法。它们基于在脱氢荧光素腺苷酸（L-AMP）存在的情况下，通过萤火虫荧光素酶（Luc，E.C. 1.13.12.7）将PPi酶促转化为ATP，随后通过两种不同方法之一测定ATP，即紫外监测（260 nm）离子对高效液相色谱法（IP-HPLC）（方法A）或在其底物萤火虫荧光素（D-LH(2)）存在下的荧光素酶依赖性生物发光法（方法B）。使用实验设计方法对这些方法进行了优化，以获得所选因素的最佳值：方法A——孵育时间（t(inc)=15分钟）、酶的失活时间（t(inac)=2分钟）、反应混合物的pH值（pH 7.50）以及L-AMP的浓度（[L-AMP]=40 microM）和荧光素酶的浓度（[Luc]=0.1 microM）；方法B——L-AMP的浓度（[L-AMP]=2 microM）、荧光素酶的浓度（[Luc]=50 nM）和荧光素的浓度（[LH(2)]=30 microM）。方法A在0.1 - 20 microM的PPi范围内具有线性响应，检测限（LOD）为0.5 microM，定量限（LOQ）为1.8 microM。以相对标准偏差（R.S.D.）表示的精密度在1 microM PPi时为7.4%，在8 microM PPi时为5.9%。方法B在0.75 - 6.0 microM的PPi范围内具有线性响应，LOD和LOQ分别为0.624和2.23 microM，在2.5 microM PPi时R.S.D.为5.1%，在5 microM PPi时R.S.D.为4.9%。在优化条件下，可以获得用于分析商业核苷酸和三聚磷酸（P(3)）中PPi杂质的灵敏且稳健的方法。
