In the preceding section, we mentioned the method of bioluminescence resonance energy transfer (BRET) and its main workhorse, luciferase. Generally, fluorescence phenomena depend on the input of energy in the form of electromagnetic radiation. However, emission of electromagnetic radiation from a system can also be achieved by prior excitation in the course of a chemical or enzymatic reaction. Such processes are summarised as luminescence . Luminometry is not strictly speaking a spectrophotometric technique, but is included here due to its importance in the life sciences.
Principles
Luminometry is the technique used to measure luminescence, which is the emission of electromagnetic radiation in the energy range of visible light as a result of a reaction. Chemiluminescence arises from the relaxation of excited electrons transitioning back to the ground state. The prior excitation occurs through a chemical reaction that yields a fluorescent product. For instance, the reaction of luminol with oxygen produces 3-aminophthalate, which possesses a fluorescence spectrum that is then observed as chemiluminescence. In other words, the chemiluminescence spectrum is the same as the fluorescence spectrum of the product of the chemical reaction.
Bioluminescence describes the same phenomenon, only the reaction leading to a fluorescent product is an enzymatic reaction. The most commonly used enzyme in this context is certainly luciferase. The light is emitted by an intermediate complex of luciferase with the substrate (‘photoprotein’). The colour of the light emitted depends on the source of the enzyme and varies between 560 nm (greenish yellow) and 620 nm (red) wavelengths. Bioluminescence is a highly sensitive method, due to the high quantum yield of the underlying reaction. Some luciferase systems work with almost 100% efficiency. For comparison, the incandescent light bulb loses about 90% of the input energy to heat. Because luminescence does not depend on any optical excitation, problems with autofluorescence in assays are eliminated.
Instrumentation
Since no electromagnetic radiation is required as a source of energy for excitation, no light source and monochromator are required. Luminometry can be performed with a rather simple set-up, where a reaction is started in a cuvette or mixing chamber, and the resulting light is detected by a photometer. In most cases, a photomultiplier tube is needed to amplify the output signal prior to recording. Also, it is fairly important to maintain strict temperature control, as all chemical, and especially enzymatic, reactions are sensitive to temperature.
Applications
Chemiluminescence
Luminol and its derivatives can undergo chemiluminescent reactions with high efficiency. For instance, enzymatically generated H2O2 may be detected by the emission of light at 430 nm wavelength in the presence of luminol and microperoxidase.
Competitive binding assays may be used to determine low concentrations of hormones, drugs and metabolites in biological fluids. These assays depend on the ability of proteins, such as antibodies and cell receptors, to bind specific ligands with high affinity. Competition between labelled and unlabelled ligand for appropriate sites on the protein occurs. If the concentration of the protein, i.e. the number of available binding sites, is known, and a limited but known concentration of labelled ligand is introduced, the concentration of unlabelled ligand can be determined under saturation conditions when all sites are occupied. Exclusive use of labelled ligand allows the determination of the concentration of the protein and thus the number of available binding sites.
During the process of phagocytosis by leukocytes, molecular oxygen is produced in its singlet state (see Figure 1 ) which exhibits chemiluminescence. The effects of pharmacological and toxicological agents on leukocytes and other phagocytic cells can be studied by monitoring this luminescence.
Fig1. Jablonski diagram. Shown are the electronic ground state ( S 0 ), two excited singlet states ( S 1 , S 2 ) and a triplet state ( T 1 ). Only select vibrational levels ( v ) are illustrated. Solid vertical lines indicate radiative transitions, dotted lines show non-radiative transitions. Inset: Explanation of total spin S and multiplicity M . The total spin S is calculated as the sum of the individual electron spins. The multiplicity M is obtained as either 1 ( singlet state) or 3 ( triplet state).
Bioluminescence
Firefly luciferase is mainly used to measure ATP concentrations. The bioluminescence assay is rapidly carried out with accuracies comparable to spectrophotometric and fluorimetric assays. However, with a detection limit of 10−15 M, and a linear range of 10−12 to 10−6 M ATP, the luciferase assay is vastly superior in terms of sensitivity. Generally, all enzymes and metabolites involved in ATP interconversion reactions may be assayed with this method, including ADP, AMP, cyclic AMP and the enzymes pyruvate kinase, adenylate kinase, phosphodiesterase, creatine kinase, hexokinase and ATP sulfurase. Other substrates include creatine phosphate, glucose, GTP, phosphoenolpyruvate and 1,3-diphosphoglycerate.
The main application of bacterial luciferase is the determination of electron transfer cofactors, such as nicotine adenine dinucleotides (and phosphates) and flavin mono nucleotides in their reduced states, for example NADH, NADPH and FMNH 2 . Similar to the firefly luciferase assays, this method can be applied to a whole range of coupled redox enzyme reaction systems. The enzymatic assays are again much more sensitive than the corresponding spectrophotometric and fluorimetric assays, and a concentration range of 10−9 to 10−12 M can be achieved. The NADPH assay is by a factor of 20 less sensitive than the NADH assay.
