Chemiluminescence and Bioluminescence, Analysis by
can be adapted for the analysis of enzymes, substrates, cofactors, inhibitors, and
metal ions, for the study of cellular processes (e.g. phagocytosis), and for toxicity
testing. Luciferase genes (
luc, lux
) are now used as reporter genes, and the gene
expression is quantitated by measuring the expressed luciferase in a BL assay.
Chemiluminescence (CL) is the light emis-
sion produced in certain chemical re-
actions. Excited singlet-state molecules
are formed in the reaction via the de-
composition of high-energy intermedi-
ates such as 1,2-dioxetanes, dioxetanones,
peroxides, or endoperoxides, which de-
cay to the ground state, and some of
the energy is released as light. The
majority of CL reactions are oxidation
reactions because these reactions can
provide sufFciently energetic intermedi-
ates for light emission (e.g. 63.5 kcal/mol
for blue light at 450 nm). One of the
best-known CL reactions is the oxida-
tion of luminol (5-amino-2,3-dihydro-1,4-
phthalazinedione): the decay of singlet
excited-state 3-aminophthalate molecules
produces a blue CL at 425 nm. Generally,
CL reactions are inefFcient in aqueous
media (CL quantum yield for luminol
0.01; i.e. 1% efFciency) but are improved
in aprotic
(e.g. dimethyl
foxide). ±igure 1 illustrates the diversity
of compounds that undergo CL react-
for movies illustrating chemiluminescent
Bioluminescence (BL) is the light emitted
from living organisms (‘‘living light’’).
Many thousands of BL species have been
identiFed (666 genera from 13 phylla),
and the Frefly (
Photinus pyralis
best-known example (Table 1). Light emis-
sion (562 nm) is produced in an ATP-
dependent oxidation of Frefly luciferin,
catalyzed by Frefly luciferase (EC
(±ig. 2). This highly efFcient reaction has
a quantum yield approaching unity (100%
efFciency). However, the majority of BL
reactions are considerably less efFcient
than the Frefly reaction. The color of
bioluminescence emissions ranges from
violet (395 nm) in the sea coral Thourella,
through blue (470 nm) in the jellyFsh
Periphylla, green (550 nm) in the glow
worm Lampyris, to red (705 nm) in the
deep-sea Fsh Malacosteus. Most BL re-
actions involve an enzyme (a luciferase)
and a substrate (a luciferin). Luciferases
and luciferins from different BL organ-
isms have different structures and com-
positions: for example, marine bacterial
luciferase is a heterodimer (40 and 37
kDa), and the luciferin is a long-chain
aldehyde, whereas Renilla luciferase is a
35 kDa monomer, and the luciferin is an
imidopyridazine derivative. Some BL or-
ganisms utilize a precharged photoprotein
instead of a luciferase; for example, in the
jellyFsh Aequorea, BL is due to the reac-
tion of calcium ions with aequorin, a stable
complex of apoaequorin and coelenter-
azine (a luciferin) that has been precharged
with an oxidant (molecular oxygen). The
BL emission is due to an energy transfer
to green fluorescent protein (G±P) from
excited-state coelenterate oxyluciferin.
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