1605 Biochemical Society Transactions ( I99 I ) I 9
Light amplification by a coupled biological system:
ATP,fintfly luciferase and recycling of Am.
t
ROGER S CHITTOCK1,CHRISTOPHER W WHARTON1,J BAZ
JACKSON1,NICHOLAS BEROVIC2 and DEREK BENYON2
1School of Biochemistry and 2School of Physics,
University of Birmingham,P 0 Box 363,Birmingham B15
2TT,uK.
As is well known the luciferase system can be used to
provide an extremely sensitive assay for ATP and this
has been widely exploited for a number of purposes.
We are studying the feasibility of devising light
amplification systems centred upon the luciferase
reaction. In order to achieve amplification it is
necesary to recycle ATP so that it can be reused by
the luciferase reaction;it is also necessary to devise
a triggering process that will lead to the release of
ATP to initiate the process.
We have investigated the recycling of ATP by using
adenylate (myokinase) and pyruvate kinases and as
well as phosphoenolpyruvate. The overall reaction
scheme is shown in Fig 1.
The scheme involves a 50% stoichiometry at the first
pass followed by recycling. The kinetic behaviour of
luciferase is unusual in that a burst of light
emission is followed by a decay to a steady state
emission. The burst occurs in ca. 300 ms and can be as
much as ten times as intense as the steady state light
emission level which is achieved in a few seconds.
Several theories which purport to explain the burst of
light emissionhave been put forward and all are based
on interaction between the two active sites of the
lOOK dimer.
We have used a fluorimeter of only modest sensitivity
and have found that peak and steady-state light output
from the luciferase catalysed reaction is linear
between 5 nH (2.5 pmoles) and 10 pH ATP. The lower
limit of detection was 5 nH in the absence of
amplification by recycling of ATP.
Initially the recycling system was tried in the
absence of added ANP but no increase in light
output as compared with the simple luciferase system
was detected. Typical concentrations of enzymes and
reagents in the recycling system were: luciferase 15
nH,luciferin 20 pN,phosphoenolpyruvate 100 pll,
myokinase 40 nH,pyruvate kinase 50 nN,HgCl2 5mtl in
25 mtl glycylglycine buffer pH 7 . 8 . The emission
monochromator of the spectrofluorimeter was set at 560
MI with a bandwidth of 15 nm.
Inclusion of ANP at 100 pN caused recycling to occur
as shown in Fig. 2; the unamplified output of light
when 100 pH ANP was added to luciferase without the
recycling components is also shown. It is notable that
the timecourse of light emission is greatly slowed in
comparison with the simple nonrecycling system. The
maximum emission occurs after ca. 2 min rather than
a few hundreds of miliseconds at 5 pll ATP.
Control experiments showed that ANP could produce a
low level of light emission in the nonrecycling system
and this was taken to result from contaminating ATP in
STOICHIOMETRY OF ATP RECYCLING
- -
FIG. 1
ZATP
ATP
+
ATP
Luci ferase
AMP
Myokinase
Pyruvdte kinose
+
PEP
50% stoichiometry on f i r s t pass then recycling
I
I
I
I
1
60
120
180
240
300
c
T i m e Is1
FIG. 2 LIGHT OUTPUT WITH AND WITHOUT RECYCLING OF ATP.
-
the ANP preparation. On this assumption the
contaminant ATP was calculated to represent 0.004% of
the ANP. The amplitude of the light emission response
with recycling where 5 nN ATP was added as a
contaminant of AHP was equivalent to an addition of 5
pN ATP to the simple system;this represents an
amplication of 1000-fold.
This level of AM contamination was not detectable in
AKP using HPLC [a gradient of 0 - 20% methanol in 0.1
n phosphate buffer was used to separate ATP andAHP1
even when the colunn was loaded to maximum capacity.
Nonetheless HPLC was used to reduce the contamination
of the ANP with ATP. The removal of ATP was not as
complete as expected and had to be carried out in the
presence of phosphate (see above) which inhibits
luciferase. A second pass through the HPLC column
using water as eluant was used to remove the phosphate
prior to assay. It was found that the ATP
contamination had been reduced to 25% of the previous
level. Interestingly the light output in a recycling
reaction was reduced by only 25% apparently showing
that increased amplification had partly compensated
for the lower level of ATP.
The requirement for the addition of excess ANP may be
rationalised in terms of the recycling Scheme (Fig.1)
in that it is necessary that myokinase 'catches' some
of the ATP that is initially present in order that it
can be recycled via ADP. The added AMP will greatly
enhance this trapping process and ensure that
recycling gets underway. The amount of added A" will
affect the time response of the system since at very
high levels the luciferase reaction will have to
'wait' until ATP is available from recycling since it
will virtually all have passed into this cycle.
Plainly there is much scope for optimisation of this
recycling process but the large number of variables
(i.e. enzymes and substrates) means that this will not
be a simple task. For the amplification to be useful
in,for example,an image storage or analysis device the
time respone will need to be improved though a useful
amplification factor of 1000-fold has already been
attained.
It may be that to improve the performance
significantly it will be necessary to further purify
all of the components of the system. Plainly as far as
A18 is concerned this will not be straightforward
since the contaminating ATP was not effectively
removed using reverse phase HPLC. Possibly ion
exchange will prove more effective and ANP should at
least be stable once it has been purified.
In conclusion it has been shown that a coupled system
coinprising several enzymes cann achieve amplification
of ATP and that this results in enhanced light output
and that although the time response is slow high
levels amplification can be achieved.