An Optimized Firefly Luciferase Bioluminescent Assay for the Analysis of Free Fatty Acids.

A firefly luciferase (LUC)-based bioluminescent assay for total free fatty acids (FFA) is presented. It is based on LUC's capability of converting FFA into fatty acyl-adenylates with consumption of adenosine 5'-triphosphate (ATP). Since ATP is a cosubstrate in LUC's bioluminescent reaction, together with firefly D-luciferin (D-LH2 ) and atmospheric oxygen (O2 ), any reduction in the assay's ATP content will lead to a decrease in the bioluminescent signal, which is proportional to the amount of FFA. Using FFA mixtures containing myristic (14:0), palmitic (16:0), stearic (18:0), oleic (18:1) and arachidonic acid (20:4) in ethanol, the assay was optimized through statistical experimental design methodology, namely fractional factorial (screening) and central composite (optimization) designs. The optimized method requires 2 µL of sample per tube in a final reaction volume of 50 µL. It is linear in the concentration range from 1 to 20 µm, with limits of detection (LOD) and quantitation (LOQ) of 1.3 and 4.5 µm, respectively. The method proved to be simple to perform, demands low reagent volumes, it is sensitive and robust and may be adapted to high-throughput screening.

LC-MS and microscale NMR analysis of luciferin-related compounds from the bioluminescent earthworm Fridericia heliota.

This paper presents the main results of RP-HPLC-MS and microscale NMR analysis performed on Accompanying similar to Luciferin (AsLn(x)), compounds present in extracts of the bioluminescent earthworm Fridericia heliota that display similarities with Fridericia's luciferin, the substrate of the bioluminescent reaction. Three isomers of AsLn were discovered, AsLn(1), AsLn(2) and AsLn(3), all of which present a molecular weight of 529 Da. Their UV-Vis absorption spectra show maxima at 235 nm for AsLn(1), 238 and 295 nm for AsLn(2) and 241 and 295nm for AsLn(3). MS(n) fragmentation patterns suggest the existence of carboxylic acid and hydroxyl moieties, and possibly chemical groups found in other luciferins like pterin or benzothiazole. The major isomer, AsLn(2), presents an aromatic ring and alkene and alkyl moieties. These luciferin-like compounds can be used as models that could give further insights into the structure of this newly discovered luciferin.

Firefly bioluminescence: a mechanistic approach of luciferase catalyzed reactions.

Luciferase is a general term for enzymes catalyzing visible light emission by living organisms (bioluminescence). The studies carried out with Photinus pyralis (firefly) luciferase allowed the discovery of the reaction leading to light production. It can be regarded as a two-step process: the first corresponds to the reaction of luciferase's substrate, luciferin (LH(2)), with ATP-Mg(2+) generating inorganic pyrophosphate and an intermediate luciferyl-adenylate (LH(2)-AMP); the second is the oxidation and decarboxylation of LH(2)-AMP to oxyluciferin, the light emitter, producing CO(2), AMP, and photons of yellow-green light (550- 570 nm). In a dark reaction LH(2)-AMP is oxidized to dehydroluciferyl-adenylate (L-AMP). Luciferase also shows acyl-coenzyme A synthetase activity, which leads to the formation of dehydroluciferyl-coenzyme A (L-CoA), luciferyl-coenzyme A (LH(2)-CoA), and fatty acyl-CoAs. Moreover luciferase catalyzes the synthesis of dinucleoside polyphosphates from nucleosides with at least a 3'-phosphate chain plus an intact terminal pyrophosphate moiety. The LH(2) stereospecificity is a particular feature of the bioluminescent reaction where each isomer, D-LH(2) or L-LH(2), has a specific function. Practical applications of the luciferase system, either in its native form or with engineered proteins, encloses the analytical assay of metabolites like ATP and molecular biology studies with luc as a reporter gene, including the most recent and increasing field of bioimaging.

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

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)).

An optimized luciferase bioluminescent assay for coenzyme A.

A new bioluminescent method for coenzyme A (CoA) quantification is described. It is based on the enzymatic conversion of dehydroluciferyl-adenylate (L-AMP) into dehydroluciferyl-coenzyme A (L-CoA) by firefly luciferase (E.C. 1.13.12.7) (LUC), which causes a flash of light that can be measured in a luminometer. The method was subjected to optimization using experimental design methodologies to obtain optimum values for the concentrations of L-AMP ([L-AMP]), luciferase ([LUC]), ATP ([ATP]) and luciferin ([LH(2)]). This method has a linear response over the range of 0.25-4 microM of CoA, with a limit of detection (LOD) of 0.24 microM and a limit of quantification (LOQ) of 0.80 microM. The assay has a relative standard deviation of about 7%. By coupling this optimized procedure to bioluminescent detection, a sensible and robust method can be obtained for the analysis of CoA.