532
Chemiluminescence and Bioluminescence, Analysis by
limit ca. 1
×
10
5
molecules). More re-
cently, the luciferase detection reaction
has been improved so that a constant light
emission is obtained. This is achieved by
adding to the assay mixture substances
(
e
.
g
.c
o
e
n
z
ym
eA
,i
n
o
r
g
a
n
i
cp
y
r
o
p
h
o
s
-
phate, oxidized cytidine triphosphate) that
minimize inhibition of the luciferase by
oxyluciferin product molecules. The gene
for Vargula luciferase, apoaequorin, and
the fused marine bacterial luciferase
luxA
and
luxB
genes are also useful as reporter
genes. Other alternatives include the CL
assay (adamantyl 1,2-dioxetane aryl galac-
toside substrate, Fig. 7) of
β
-galactosidase
expressed by the
lacZ
gene and the BL
assay (±refly luciferin-
O
-phosphate sub-
strate, Fig. 7) of the enzyme expressed
by the alkaline phosphatase gene. BL
genes are also effective for
in vivo
non-
invasive imaging of whole organisms (e.g.
plants, animals).
Membrane-permeable luciferin deriva-
tives such as ethyl, hydroxyethyl, and
S
N
S
N
COOR
H
HO
1, R
=
CH
2
OH
CH
2
2, R
=
CH
2
CH
2
CH
2
OH
3, R
=
CH
3
CH
2
S
N
S
N
COOH
H
O
3
PO
4
O
O
OMe
b
-
D-galactopyranoside
5
Fig. 7
Structure of three
membrane-permeable Frefly luciferin
esters (1, hydroxyethyl; 2, hydroxypropyl;
3, ethyl), (4) bioluminescent alkaline
phosphatase substrate Frefly
luciferin-
O
-phosphate, and (5) the
chemiluminescent galactosidase
substrate.
hydroxypropyl esters (Fig. 7) permit non-
destructive measurement of ±refly lu-
ciferase reporter gene expression. These
luciferin esters cross the cell membrane
and react with intracellular esterases to
produce luciferase. This enzyme then re-
acts with ±refly luciferase to give light; the
unreacted ester is not a substrate for the
luciferase and does not contribute to the
signal. Although not a BL reporter gene,
the increasingly popular green fluorescent
protein (GFP) is derived from a BL or-
ganism, the jelly±sh
Aequorea victoria
.The
advantage of this reporter is that the highly
fluorescent expressed GFP can be detected
directly using a suitable excitation light (ca.
470 nm) without the need to add reagent
or disrupt the cells.
4.5
Rapid Microbiology
ATP (adenosine 5-triphosphate) is present
in all living cells – a bacterium contains on
average 1 fg of ATP – and measurement
of ATP using the ±refly luciferase reaction
is a convenient means of enumerating
cells, with a sensitivity of 10
5
colony-
forming units per milliliter. The procedure
entails ±rst destruction of somatic ATP
using the enzyme apyrase (EC 3.6.1.5),
followed by lysis of intact cells with
a detergent to release ATP, and ±nally
BL assay of the released ATP using
a mixture containing ±refly luciferase,
luciferin, and magnesium ions in the
appropriate buffer.
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