Bioluminescence enzymes

Seliger, H. H., and McElroy, W. D. (1964). The colors of firefly bioluminescence enzyme configuration and species specificity. Proc. Natl. Acad. Sci. USA 52 75-81. 

Gautier S.M., Blum L.J., Coulet P.R., Fiber-optic sensor with Co-immobilized bacterial bioluminescence enzymes, Biosensors 1989 4 181. 

Chemical immobilization procedures of bioluminescent enzymes such as firefly luciferase and bacterial luciferase-NAD(P)H FMN oxidoreductase to glass beads or rods [174, 175], sepharose particles [176], and cellophane films [177] have produced active immobilized enzymes. Picomole-femtomole amounts of ATP or NAD(P)H could be detected using immobilized firefly luciferase or bacterial luciferase-oxidoreductase, respectively. 

The second one contained the bacterial bioluminescent enzymes. This system made it possible to reach a detection limit of 0.5 [imol/L. 

Table 8 Analytical Performance of the Immobilized Bioluminescent Enzymes...

The first insoluble derivatives of bioluminescent enzymes were prepared by Erlanger et al. by reacting luciferases they investigated the properties of these immobilized enzyme preparations and their potential for studying the mechanism of bioluminescence [54]. 

Kurkijarvi et al. were the first to demonstrate the feasibility of seg-mented-flow bioluminescence assays by use of a bioreactor packed with bacterial bioluminescent enzymes immobilized on Sepharose 4B [60]. The packed glass colunrn used was placed in front of the photomultiplier tube of a luminometer. The luminescence signal obtained was linearly related to the NADH concentration from 1 pmol to 10 nmol for sample volumes of 2-20 pL. In the region of 400 NADH assays could be performed with a single enzyme column, with no appreciable change in sensitivity or accuracy. However, problems arising from packing or disruption of the matrix were encountered after 4 days of intensive use. 

Ito, K. Nakagawa, K. Murakami, S. Arakawa, H. Maeda, M. Highly sensitive simultaneous bioluminescent measurement of acetate kinase and pyruvate phosphate dikinase activities using a firefly luciferase-luciferin reaction and its application to a tandem bioluminescent enzyme immunoassay. Anal. Sci., 19, 105-109 (2003)... 

Quantum Yield Efficiency of fluorescence percentage of incident energy emitted after absorption. The higher the quantum yield, the greater the intensity of the fluorescence, luminescence, or phosphorescence. See Papp, S. and Vanderkooi, J.M., Tryptophan phosphorescence at room temperature as a tool to study protein structure and dynamics, Photochem. Photobiol. 49, 775-784, 1989 Plasek, J. and Sigler, K Slow fluorescent indicators of membrane potential a survey of different approaches to probe response analysis, J. Photochem. Photobiol. 33, 101-124, 1996 Vladimirov, Y.A., Free radicals in primary photobiological processes, Membr. Cell Biol. 12, 645-663, 1998 Maeda, M., New label enzymes for bioluminescent enzyme immunoassay, J. Pharm. Biomed. Anal. 30, 1725-1734, 2003 Imahori, H., Porphyrin-fullerene linked systems as artificial photosynthetic mimics, Org. Biomol. Chem. 2, 1425-1433, 2004 Katerinopoulos, H.E., The coumarin moiety as chromophore of fluorescent ion indicators in biological systems, Curr. Pharm. Des. 10, 3835-3852, 2004. 

Bioluminescent enzyme systems based on bacterial and firefly luciferases offer a unique and general tool for analysis of the many analytes and enzymes in the environment, research and clinical laboratories and other fields. 

However, native bioluminescent enzymes are generally subject to inactivation in vitro, and hence not suitable for routine analytical use. Immobilized enzymes and whole bacteria largely solve this instability problem, and hence enable the routine use of bioluminescent analysis with high speed, specificity, simplicity, sensitivity and accuracy. Inunobilization also enables the development of automated luminescent biosensors. 

Fukoda S, Tatsumi H, Maeda M. Bioluminescent enzyme immunoassay with biotinylated firefly luciferase. J Clin Ligand Assay 1998 21 358-362. 

DEVELOPMENT OF TANDEM BIOLUMINESCENT ENZYME IMMUNOASSAY FOR ANGIOTENSIN I AND ENDOTHELIN-1... 

We developed a simultaneous bioluminescent assay of acetate kinase (AK) and pyruvate phosphate dikinase (PPDK). In the method, the detection limits (blank + 3SD) of AK and PPDK were 1.03x10 and 2.05x10 mol/assay, respectively. Previously, we successfully applied a tandem bioluminescent enzyme immunoassay (BL-EIA) for simultaneous measurement of insulin and c-peptide. In this study, we also applied the method to tandem BL-EIA for Angiotensin I and Endothelin-1, which are hypertension related peptides. The tandem BL-EIA used a competitive immuno-reaction for Angiotensin I and sandwich inununo-reaction for Endothelin-1. 

To estimate the contribution of the second mechanism the interaction of bioluminescent enzymes with halide-containing dyes was investigated. Two types of dyes were tested xanthene and anthracene derivatives. The interaction of dyes with bacterial luciferase and apo-obelin was studied. 

Bioluminescent enzymes bacterial luciferase from Photobacterium leiognathi and apo-obelin from the marine hydroid polyp Obelia longissima were purchased from the Photobiology Laboratory (Institute of Biophysics, SB RAS, Krasnoyarsk, Russia). Anthracene derivatives (2-chloranthracene (CA), 9-bromanthracene (BA)... 

Thus, the interaction of bioluminescent enzymes with haloid-containing dyes was found and characterized. From results obtaines can be concluded that the contribution of the biochemical mechanism (i.e. the action of the heavy atom on the catalytic activity of the enzyme) is much greater than that of the physicochemical mechanism. 

K. Kurkijarvi, T. Heinonen, T. Lovgren, J. Lavi, and R. Raunio, Flow-Injection Analysis with Immobilized Bacterial Bioluminescence Enzymes. Anal. Appl. Biolumin. Chemilumin., 3rd (1984) 125. 

Roda, A., Girotti, S., Ghini, S., Carrea, G., Continuous-Flow Assays with Nylon TUbe-Im-mobilized Bioluminescent Enzymes , in Methods in Enzymology Vol. 137, Colowick, S. P., Kaplan, N. O., Mosbach, K. (eds) San Diego Academic Press, 1988, pp. 161-171. 

Marine bacterial luciferase can be immobilized on to a variety of solid supports (e.g., Sepharose, nylon) and it has also been coimmobilized with NAD(P)H FMN oxidoreductase. The close proximity of the two enzymes leads to efficient channeling of FMNH2 produced by the oxidoreductase to the luciferase. More extensive coupled coimmobilized systems have been prepared in which all of the enzymes in the enzymatic conversion of glucose to alcohol were coimmobilized with bacterial luciferase and the oxidoreductase, and the bioluminescent enzymes were used to monitor different stages in the biotransformation of glucose. 

The first assay developed by one of us was for primary bile acids using cy-HSD and bioluminescent enzymes immobilized on Sepharose 4B (Table 4). A linear response in the range 1.5 to 50 mol/l was obtained for SBA on serum heated at 70 °C for 15 min. The use of a Sepharose-immobilized enzyme system increases the catalytic activity per unit enzyme and gives more stable analytical reagents. 

The 12o(-HSD, a NADP" dependent enzyme, was so coimmobilized with bioluminescent enzymes on Sepharose 4B. To improve sensitivity bacterial diaphorase was sometimes used instead NADPHrFMN oxidoreductase, since background is less. The diaphorase containing systems are very sensitive to differences in mixing and shaking. However with an injection method the inter -assay precision is good (8%) and the standard curve is linear from 0.4 to 200 mol/1. 

Bioluminescent Enzyme Immune Assay (BLEIA), where the enzyme is the marker for the antigen of the immune reaction, which results in light emission. Markers are enzymes that are conjugated to bacterial luciferase, such as peroxidases, acid phosphatases and dehydrogenases. 

Bioluminescent Enzyme Multiproductive System (BLEMS), in whieh enzymatic luminescent immune reactions are triggered. ATP and NAD(P)H are the final products, which are tested by firefly- and bacterial luciferase. Here, conjugates of the substrate of interest to one of the BL reagents are used. 

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