Chemistry Reference
In-Depth Information
Luciferase, O
2
Luciferin
Oxyluciferins +
ATP
AMP
fIgure 11.38
The general mechanism for bioluminescence.
the oxidation of luciferin. In some cases, luciferin is a protein known as photoprotein, which upon binding to metal ions,
such as calcium(II) ions or magnesium(II) ions, will activate the reaction during a light-making process. Such binding to
co-factors leads to a conformational change of the protein, giving the living organisms a way to precisely control the light
production and emission. Adenosine triphosphates (ATP) are often consumed during the processes. Of course, there are also
neurological, mechanical, chemical, or as-yet-undiscovered triggers that can start the reactions to release energy in form of
light (Figure 11.38) [168]. In bacteria, luminescence involves the oxidation of FMNH
2
along with a long-chain aldehyde
and a two-subunit luciferase, which is controlled by an operon called Lux operon.
Over the last decade, bioluminescence imaging has become a powerful methodology for molecular imaging of small
laboratory animals, which allows the study of ongoing biological processes
in vivo
. This can be achieved by detecting lucif-
erase in genetically modified cells, bacteria, or animals. Although bioluminescent probes (luciferases) are not so popular
because they are exceedingly dim in comparison with fluorescence, their unique and advantageous features render them
promising for wide applications. For example, these probes do not require exogenous illumination, and there is no photo-
toxicity or photobleaching of the emitting molecules or artificial perturbations of light-sensitive cells. Even though biolu-
minescence is much dimmer, it can be up to 50 times more sensitive than fluorescence, and images can be generated with
remarkably high signal-to-noise ratios because there are low background noises, with the tissues having rather low intrinsic
bioluminescence [169]. The reasons for such low background are that compared with endogenous fluorescence (autofluo-
rescence), which can be as bright as the signal itself, endogenous bioluminescence (autoluminescence) of most cells is
extremely low, and that there are no excitation photons that contribute greatly to background in fluorescence imaging.
Therefore, the background levels in bioluminescence imaging are especially low and the signal-to-noise ratio can be very
high, although the signals are very dim.
Biological events with fast kinetics can also be detected by adapted instruments, where
in vitro
luminescence inten-
sities are quantitative to the amount of labelled cells. However, the absence of background signals in bioluminescence
makes the localisation of the contour of the animal impossible, and imaging on moving animals requires an additional
tracking system to follow their motion. until recently, bioluminescence imaging has been harnessed to detect ATP
because the luciferase reaction depends on the presence of oxygen and ATP, and this has had a much larger range of
applications. Luciferase activity can now be used to detect protein-protein interactions, tracking cells
in vivo
, measuring
levels of calcium and other signalling molecules, detecting protease activity, and even reporting circadian clock gene
expression [170].
Among many luminescent species existing in nature, only three luciferases have been studied in detail and are being
used in biomedical research: the
Photinus pyralis
(firefly) luciferase (Fluc), the sea pansy
Renilla reniformis
luciferase
(rluc), and the marine copepod
Gaussia princeps
(Gluc) (Figure 11.39). Gluc and rluc give off blue luminescence
(480 nm), which is strongly absorbed by pigmented molecules such as hemoglobin and melanin. However, blue emission
is easily scattered by tissues and hence makes them less suitable for
in vivo
imaging. In contrast, Fluc is a better candidate
for
in vivo
imaging, because it emits green light (562 nm). The sensitivity of bioluminescence imaging in deep tissues can
be enhanced by the use of red emitting luciferases from
Pyrophorus plagiophthalamus, Lhotinus pyralis, Luciola italic,
and the railroad worm. Firefly luciferase was first cloned in 1985 and soon it had developed into an assay to measure
luciferase in mammalian cell lysates. This development enabled the luciferase gene to become a useful tool for
in vivo
studies of gene regulation [171].
Bioluminescence has become indispensable for noninvasive monitoring of biologic phenomena
in vitro/vivo
by various approaches, such as genetic receptor assays (measure changes in luciferase production), ATP assays (mea-
sure changes in ATP levels) and Luciferin Substrate Assays (measure enzymatic release of free luciferin). Inside the
nucleus, bioluminescence is available to monitor mirNA expression and evaluate sirNA delivery to specific tissue by
fusing luciferase to the mirNA target site at the 3′ uTr and silencing sirNA of luciferase expression respectively.
In the cytoplasm, protein folding and secretion can be monitored by chaperone-mediated protein-folding using secreted
luciferase [171].
