103
A LABORA TORY EXPERIMENT ON PURIFICATION AND
CHARACTERIZATION O F F I R E F L Y L U C I F E R A S E
JUDY A HALL, JoANN J WEBSTER and FRANKLIN R LEACH
D e p a r t m e n t of Biochemistry
O k l a h o m a State University
Stillwater, O k l a h o m a 74078, U S A
Introduction
For the past 3 years in a biochemistry laboratory course for graduate student majors we
have purified and characterized firefly luciferase. Since the students obtained good results
and enjoyed the experiment, we now report it for others to use. The purification scheme is
an unpublished modification and combination of methods. Illustrative data are presented
to show typical results and to allow the instructor to select the appropriate parts of the
experiment for the class.
Suelter I has evaluated some of the questions often asked about the purpose of and
possible approaches to the biochemistry laboratory experience. Since preparation of
reagents and development of the proper protocol are basic to proper experimentation,
these possibilities should be considered when developing a laboratory experiment.
Enough detail is given in the current description for the instructor to operate with either a
structured format or the open format described by Suelter. The choice depends on the
time available, the students, and the goals of the instructor for the class.
Background
Firefly luciferase (EC No 1.13.12.7) catalyzes the reaction shown in Fig. 1. This reaction is
a means of communication between the sexes before mating. Lloyd 2 has recently reviewed
the role of bioluminescence for communication in insects. The males of the grassland
firefly of Florida (Photinus collustrans) begin their evening search for a mate about 21 min
after sunset. At first the altitude of their search is below 1 m but they climb a little when it
gets darker. The females leave their underground burrows about the same time. The
'average' male spends more than seven evenings trying to locate a female. The female sees
and responds to a male flash; she attracts a male, copulates, and returns underground in
usually less than 6 min.
The luciferase from Photinus pyralis has a molecular weight of 105, is comprised of two
apparently identical subunits, has a pH optimum of 7.8, has an optimum temperature of
25°C; binds two luciferins, one MgATP, two ATPs, one luciferyl adenylate, and has two
essential -SH groups. It is extremely hydrophobic, and is inhibited by dehydroluciferin,
AMP, ATP, PPi, and anions. 3
Firefly luciferase was first crystallized by Green and McElroy. 4 A simpler purification
procedure has been developed by Shimomura, Goto, and Johnson. 5 The development of
modern electronic instrumentation 6 and commercial firefly luciferase-luciferin preparations (see ref 7 for a comparison) has increased the analytical uses of this reaction. Two
symposia 8'9 devoted to ATP analysis have appeared and two international symposia l°'H
PP
Ot
,
AMP
co,
01"-LU
LU
0 L,~-.,.-...., , , ~ \
"" OL" LU
Figure 1
The firefly luciferase reaction scheme. LU is the firefly luciferase, LH2 is luciferin,
LHe.AMP is luciferyl adenylate, OL* is activated oxyluciferin, and OL is oxyluciferin.
There is no required order of addition of LH2 and MgA TP
BIOCHEMICAL EDUCATION 12(3) 1984
104
on the analytical applications of bioluminescence and chemiluminescence have been held.
Leach 12 has reviewed the use of firefly luciferase for ATP determination.
McElroy 13 obtained a standard curve for the measurement of ATP in amounts ranging
from 10 to 150 ~g visually by observing the time required for the disappearance of light
from mixtures of luciferase and ATP. This gives departments which do not have
photometers or scintillation counters a means of measuring firefly luciferase.
Luciferase purification
(Modified from refs 5, 14, and 15) An outline of this purification scheme is shown in Fig. 2.
The students work in groups of 2 or 3.
Firefly Tails, 59
I
Grind
150 ml Iocetone
I
5 0 ml acetone
I
I
Dry overnight
I
Discard
I
Grind in buffer
I
Centrifuge
15K 3 0 m i n
I
II
Discard
!
(Crude Extract)
I
[NH4)z SO4
I
II
0-035
II
035-075
I
Discard
I
Discard
D i ss o~ve ( (N H 4)2 SO4 Fraction)
I
Chromatograph
on
Sephadex G- 150
I
Combine
Fraction with activity (Sephadex Fraction)
I
I
0-07
Crystallize
Figure 2
( NH4 )2 S04
I
i
Discord
(Crystals)
Purification scheme. The procedure used to purify firefly luciferase is shown in outline
form. Precipitates are shown to the left of the steps and are indicated by two vertical lines.
The supernatant solutions are on the right and are indicated by a single vertical line
(1) Acetone powder Grind 5 g of dried firefly lanterns (Sigma product number FFI') in the
coldroom with a mortar and pestle to a fine powder. Add the dry powder to 150 ml of cold
acetone, gently swirl, and allow to stand in the cold for 30 min. Filter on a Buchner funnel,
wash once with 50 ml of cold acetone, and then maintain suction until the cake is dry and
powdery. Usually 15 min of aspiration in the cold is adequate. Store overnight in a
desiccator. Done on day 1.
(2) Extraction Grind the acetone powder in a mortar and pestle with 50 ml of 25 mM
Tricine buffer containing 5 mM MgSO4 and 1 mM EDTA at pH 7.8. The pH must be
maintained above pH 7.5 by addition of 1 M NaOH as required (a drop of phenol red and
a drop of the solution to be tested in a 6 x 50 mm tube gives a pinkish-orange to purple
color and indicates a pH greater than 7.5). Centrifuge at 27 000 × g for 30 min in the cold.
Save a sample and label it crude extract.
(3) Ammonium sulfate fractionation Add 0.7 volume of a solution 80% saturated with
(NH4)2SO4 (516 g + 1 i of water) at pH 7.8 dropwise to the supernatant solution.
Centrifuge as before for 30 min. Slowly add 0.28 g of ground solid (NH4)2SO4 per ml of
the supernatant solution again keeping the pH above 7.5. Centrifuge as before and retain
the precipitate for the next step.
(4) Gel filtration on Sephadex G-150 Dissolve the precipitate in about 5 ml of 10 mM
Tricine buffer containing 2 mM EDTA and 10% (NH4)2SO4, pH 7.8 (save a sample and
label (NH4)2S04fraction). Gel filtration takes place on a Sephadex G-150 column (2.2 ×
70 cm) equilibrated with the above buffer. Fractions of 5 ml are collected, the protein
content is determined as A28onm, and firefly luciferase activity is determined. Fig. 3 shows
typical results. The gel chromatography runs overnight of day 2.
BIOCHEMICAL EDUCATION 12(3) 1984
105
i
!
I
i
•
i
1.0
10
i.
I?,:
2
Figure 3
0
Fraction
I1
n
i
S
i
'
i
'
0
Number
Gel filtration of firefly luciferase. The elution of firefly luciferase from a Sephadex G-150
column is shown. The protein, o, was measured as A2so and the enzyme activity, o, was
determined as described in the text using a 30-s assay time
(5)Crystallization The fractions containing significant luciferase activity are combined
(save a sample and label column fraction). The combined fractions are treated with 0.47 g
(NH4)2SO 4 per ml of solution. After centrifugation the precipitate is dissolved in a
minimum volume of 10% (NH4)2SO4 containing 1 mM EDTA at pH 8. Dialyze overnight
against 1 1 of 1 mM EDTA, 10 mM NaC1, and 2 mM Na2HPO4 at pH 7.3 using one buffer
change. This is day 3. Harvest the crystals by centrifugation and dissolve in 10%
(NH4)2SO4 containing 1 mM EDTA pH 8. Save a sample and label crystals. As soon as the
activity has been determined, bovine serum albumin is added to stabilize the enzyme (100
Ixg ml-]). The stabilized enzyme preparation may be stored frozen for 10 days.
Experiments
(Day 4)
(1) Enzyme assay 16 The assay system contains: 25 mM Tricine buffer, pH 7.8, 5 mM
MgSO4, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 Ixg luciferin (Sigma # L 6882), 100 I~g
bovine serum albumin, and an appropriate amount of luciferase. The total volume is
usually 1 ml but may be reduced to 0.5 or 0.2 ml depending on the photometer used. The
reaction is initiated by the addition of 1 ng of ATP or by the addition of the luciferase.
Because of the effects of geometry and volume changes on the measurement of light
production and since no standardized unit of luciferase activity has been defined, the
results are reported in light units which is the production of 103 counts in a defined time
interval (30 or 60 s) in the instrument used.
(2) Purification table See Table 1 as an example. Measure the volume, protein
concentration (A280 of 0.75 in a 1-cm cell equals 1 mg mll: see ref 14) and enzymatic
activity. Save all fractions until the activity is located.
(3) Proportionality Demonstrate the proportionality of light production to enzyme
concentration by varying the amount of luciferase in the reaction mixture while all other
factors are kept constant. Figure 4 shows typical results.
Table 1
Summary of firefly luciferase purification
Fraction
Volume
(ml)
Crude extract
52
7.5
118
10
(NH)4)2SO4
Column
Crystals
Protein
total
(mg/ml) (g)
Activity
Specific Total
Light units
per mg (xlO -3)
493
91
2.5
2.9
135
2324
3954
21 830
25.6
0.684
0.295
0.029
3456
1598
1166
633
Yield
(%)
46
34
18
Purification
1
17
29
162
The purification was by the outlined procedure. Protein was determined at each step by .428o measurement,
and the luciferase activity as described in the Methods. The production of 1 x 103 counts in the defined assay
time is a Light unit. With the crude extract fraction there is light scattering and other substances which
contributes to the anomalous value observed.
BIOCHEMICAL EDUCATION 12(3) 1984
106
Li 400
h
I
g
I1
i 20O
t
/o
/
g
/
g~
/
'
2.~9
ng
Figure 4
'
s.~a
Luciferase
Proportionality of light output to protein concentration. Various concentrations of luciferase
are used in the standard assay. The amount of luciferase is multiplied by 10 -3, ie ~g instead
of ng
(4) Assay components Determine the effect of omission of each of the assay components.
Table 2 contains typical results.
Table 2
Effect of omissions from the assay mixture on luciferase activity
Component omitted
Light production, light units
None
MgSO 4
EDTA
Dithiothreitol
Luciferin
Bovine serum albumin
ATP
84.0
0
67.4
81.4
0
64.2
1.5
The concentration of each reaction component is given in the text. A
light unit is 1000 counts per min produced with 1 ng of ATP.
(5) K,,, Determine the Km for luciferin by varying the amount of luciferin added in the
assay. A typical saturation curve is shown in Fig. 5.
300
/ ~ o - -
t
i
0
hi
°
fl
i
I
oo.o
200
/(
/''
/
oo~
/o
f
i
~s
I
50
Figure 5
I
ot
I
pg
100
i
.
150-"
200
Luciferin
Effect of luciferin concentration. The crystalline firefly luciferase was supplemented with the
indicated concentration of Sigma luciferin and the light output was determined
(6) Standard curve Construct a standard curve for ATP and determine the amount of ATP
present in the unknown sample given to each student. Because of the range of response,
the plot is made on log-log paper (see Fig. 6 for an example).
(7) Kinetics Follow the kinetics of the reaction. Firefly luciferase displays various types of
kinetics depending upon the conditions used. ~7 We find that rapid-reaction kinetics
depend upon the concentration of ATP used. With 0.2 ng of ATP there is an almost
BIOCHEMICAL EDUCATION 12(3) 1984
107
constant output of light over a few minutes while with 200 ng a flash of light is produced,
followed by a decay in light production. Figure 7 shows these types of kinetics as
determined using a recorder and rapid injection of ATP.
500
'
L 100
li
I
I
'|i
$
://
'/
'
/
/zr
1,O
I
l
10
Figure 6
l
I
I
1 O0
pg ATP
I
1000
Effect of A TP concentration. The effect of various concentrations of A TP on light output is
shown
10
I
i
I
'
0.4
I
0.2ng
8
0.3
mV
mV 6
0.2
4
0.1
2
I
|
Figure 7
I
30
I
Seconds
I
30
60
m
60
Seconds
Kinetics of firefly luciferase using two concentrations of A TP. The reaction was initiated by
the injection of A T P and was followed on a Houston Instruments Omni-Scribe recorder.
Voltage output is plotted against time
Acknowledgments
This manuscript (J-4336 of the Oklahoma Agricultural Experiment Station) was read by Drs O C Definer, M K
Essenberg, I D Eubanks, R E Koeppe and E C Nelson. The work was supported in part by project number 2103
of the Oklahoma Agricultural Experiment Station. I thank the students who have tested the method and made
suggestions for improvement.
References
i Suelter C H (1982) Biochem Educ 10, 18-21
2 Lloyd J E (1983) Ann Rev Entomol 28, 131-160
3 DeLuca M A (1976) Adv Enzymo144, 37-68
4 Green A A and McElroy W D (1956) Biochim Biophys Acta 20, 170-176
5 Shimomura O, Goto T and Johnson F H (1977) Proc Nat Acad Sci USA 74, 2799-2802
6 Stanley P E (1982) in Clinical and Biochemical Luminescence (Kricka L J and Carter T J N, Editors) Dekker,
New York
7 Webster J J, Chang J C, Howard J L and Leach F R (1979) J Appl Biochem 1,471-478
8 Borun G A (Editor) (1975) Proceedings of ATP Methodology Seminar, SAI Technology Co, San Diego, CA
9 Borun G A (Editor) (1977) Proceedings of the Second Bi-Annual ATP Methodology Symposium, SAI
Technology Co, San Diego, CA
~0 Schram E and Stanley P E (Editors) (1979) International Symposium on Analytical Applications o]
Bioluminescence and Chemiluminescence, State Printing and Publishing, Inc, Westlake Village, CA
t l DeLuca M A and McElroy W D (Editors) (1981) Bioluminescence and Chemiluminescence, Academic Press,
New York
12 Leach F R (1981) J Appl Biochem 3, 473-517
13 McElroy W D (1947) Proc Natl Acad Sci USA 33, 342-345
14 DeLuca M A (1978) Meth Enzymol 57, 3-15
15 Howard J L Personal communication
t6 Webster J J and Leach F R (1980) J Appl Biochem 2,469-479
17 DeLuca M, Wannlund J and McEiroy W D (1979) Analyt Biochem 95, 194-198
B I O C H E M I C A L E D U C A T I O N 12(3) 1984