Analytical Chemiluminescence/Lucigenin and coelenterazine
B4. Lucigenin and coelenterazine
Lucigenin and related 9,9/-diacridinium salts give an intense blue-green emission when oxidized by alkaline hydrogen peroxide. The major chemiluminescence emitter is postulated to be N-methyl acridone (blue light), produced via a peroxide, with other excited molecules involved. The reaction is catalysed by pyridine, piperidine, ammonia or osmium tetroxide. A proposed mechanism explains the chemiluminescence of oxidized acridinium salts by the formation of excited peroxide intermediates.
Lucigenin is used in a wide variety of assays, especially those involving enzymatic production of hydrogen peroxide, and as a label in immunoassays. It reacts with various reductants, including those present in normal human blood, such as glutathione, uric acid, glucuronic acid, creatinine, ascorbic acid and creatine. The chemiluminescence intensity for a mixture of these analytes is equal to the sum of the intensities, measured separately for each analyte present. Metal ions – iron(III), manganese(II) and copper(II) – also contribute to the chemiluminescence and so must be regarded as interferents. Lucigenin is also affected by a very wide range of other metal ions, both enhancers and inhibitors. The most effective enhancers are osmium (VIII), cobalt(II), ruthenium(III), iron(II) and iron(III) and the most effective inhibitors are europium(III), thorium(IV), ytterbium(III), terbium(III) and manganese(II). Among the enhancers, effective enhancement seems to be associated with low detection limit but this association is much less pronounced among the inhibitors.
Lucigenin chemiluminescence has been important for the determination of superoxide. The mechanism for the lucigenin-superoxide reaction is believed to be:
(B4.1) Reduction to cation radical: Luc2+ + e- → Luc•+
(B4.2) Coupling to yield dioxetane: Luc•+ + O2•– → LucO2
(B4.3) Decomposition of dioxetane to N-methylacridone: LucO2 → NMA* + NMA
(B4.4) Chemiluminescence: NMA* → NMA + light
The credibility of lucigenin detection of superoxide has been questioned because of the evidence (disputed) for a process called redox cycling in which lucigenin reacts with oxygen to form more superoxide, leading to the amount of superoxide being overestimated. As a result, coelenterazine (a luminophore from the coelenterate Aequorea), became a more favoured probe for superoxide; although this also offered improved selectivity for superoxide, it was not entirely specific. Attention has therefore shifted to assays using Cypridina luciferin analogues (see chapter B3) to detect superoxide.
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