The sterilization of the protein fruitfly luciferase under conditions that prevent denaturation is examined. Denaturation is prevented by heating the protein in contact with molecular seives and under a reduced pressure of the order of 0.00005 millimeters of mercury

Chappelle, E. W.

Rich, E., Jr.

English

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United States Patent Office 3,745,089Patented July 10, 1973
3,745,089
PROTEIN STERILIZATION METHOD OF FIREFLY
LUCIFERASE USING REDUCED PRESSURE AND
MOLECULAR SIEVES
Emmett W. Chappelle. Baltimore, and Edward Rich, Jr.,
College Park, Md., assignors to the United States of
America as represented by the Administrator of the
National Aeronautics and Space Administration
No Drawing. Filed Mar. 5, 1968, Ser. No. 710,621
Int. Cl. C07g 7/02
U.S. Cl. 195—66 R 5 Claims
ABSTRACT OF THE DISCLOSURE
Denaturation of the protein firefly luciferase during
sterilization thereof is prevented by heating the protein in
contact with certain molecular sieves, and under a reduced
pressure of the order of 5x 10~* millimeters of mercury.
The invention herein described was made by employees
of the United States Government and may be manufac-
tured and used by or for the Government for govern-
mental purposes without the payment of any royalties
thereon or therefor.
This invention relates to the sterilization of the protein
firefly luciferase, and more particularly to the sterilization
of firefly luciferase under conditions which prevent the
denaturation of this protein. Specifically, the invention re-
lates to sterilization of firefly luciferase in contact with
certain molecular sieves, and under a high vacuum, where-
by denaturation of the protein is prevented.
The utilization of luciferase in life detection techniques
has been the subject of considerable experimentation over
the past years. This research was based on the light pro-
ducing reactions of the firefly, and on the fact that all ter-
restrial life is intimately associated with, and dependent
upon, the nucleotide phosphate adenosine-triphosphate
(ATP). The bioluminescent reaction occurring in the
firefly is the result of the reaction of oxygen with an
oxidizable substrate (luciferin) catalyzed by an enzyme
(luciferase). In order for this reaction to proceed, the
luciferin must first react with the ATP before it can be
oxidized for such light production.
For life detection use, all of the ATP is first removed
from any of the components of the luciferin-luciferase
mixture, after which the mixture is placed in proximity to
the suspect area while maintaining a light fast environ-
ment. If life is present, the ATP associated therewith will
activate the mixture with the resultant light emission. This
light can be measured by light monitoring equipment well
known to those skilled in the art.
One of the difficulties of the foregoing procedure is that
the enzyme luciferase cannot be heat sterilized without
destroying its activity when in contact with ATP. Steriliza-
tion of proteins by heat has never been successfully ac-
complished because of the instability of proteins when sub-
jected to heat.
Accordingly, it is an object of the present invention to
provide a method for sterilizing the enzyme luciferase
without destroying its activity for reaction with ATP.
It is also an object of this invention to permit steriliza-
tion of luciferase by heating while retaining a substantial
proportion of the activity of luciferase for reaction with
ATP.
Other objects of the present invention are apparent from
a consideration of the following detailed description and
claims.
In general, this invention provides a method for steriliz-
ing the enzyme luciferase while preventing denaturation
5 thereof comprising heating the luciferase under a reduced
pressure, and at a temperature and for time sufficient to
sterilize same wherein the luciferase is in contact with a
molecular sieve selected from the group consisting of those
bearing the trade names Sephadex G-25, and Biogel
10 P-300. The pressure must be sufficiently low to prevent
complete loss of activity of the luciferase for reaction
with ATP.
As mentioned above, the pressure under which the
method of this invention is effected should be sufficiently
15 low to prevent complete loss activity of the enzyme
luciferase for reaction with ATP. Specifically, when the
heating of the enzyme is initiated, the pressure should not
exceed 10-* millimeters of mercury.
The luciferase should be heated for a time sufficient to
20 sterilize same; however, the time will vary with the mo-
lecular sieve used, the temperature employed, and the
pressure under which the sterilization ocurs. Specifically,
the time required to sterilize the luciferase can vary within
the range of 6 hours to 36 hours.
25 The temperature at which sterilization is effected will
vary with the other reaction conditions and with the mo-
lecular sieve employed, but will generally be within the
range of 120° to 135° centigrade.
The molecular sieves employed in the method of this
30 invention are selected from the group consisting of those
bearing the trade names Sephadex G-25 and Biogel
P-300. Sephadex G-25 is manufactured by Pharmacia
Fine Chemicals, Inc., and is a cross-linked dextran gel
with a pore size that corresponds to a protein with a
35 molecular weight of approximately 5000. Biogel P-300,
which is manufactured by Bio-Rad Laboratories, is a
polyacrylamide gel with pore size corresponding to a pro-
tein with a molecular weight of approximately 400,000.
Both of these gels are chemically inert. Biogel P series
4" gels are described more fully in price list "S," published
Feb. 1, 1967, by Bio-Rad Laboratories, at pages 39-45.
The following example shows the effectiveness of the
method of this invention in retaining the activity of firefly
luciferase for reaction with ATP after sterilization by
45 heating and contrasts the results obtained with Sephadex
G-25 and Biogel P-300 to the results obtained by sterili-
zation of firefly luciferase in contact with other specified
materials.
50 EXAMPLE
The experimental procedure was as follows: 2 ml. of
luciferase solution [containing partially purified luciferase,
1 mg. of protein/ml.; luciferin, 0.5 mg./ml.; magnesium
sulphate, 0.01 M; and tris buffer (pH 7.4), 0.05 M] was
55 added to separate tubes of Sephadex G-25, Biogel P-300,
dextran, bovine serum albumin, and diethylaminoethyl
cellulose (DEAE). These five mixtures were quick-frozen
in liquid nitrogen and lyophilized for 24 hours. Then, in
replicates of three, the following treatments were imposed:
60 (1) Storage at —80° C. for 36 hours;
(2) Exposure in air at 135° C. for 36 hours;
(3) Exposure to 135° C. for 36 hours with an initial
chamber pressure of 5x 10-4 mm. of mercury which rose
to 4x 10~3 mm. of mercury during heating.
65 After these treatments, samples were prepared for assay
by the addition of 5 ml. of distilled water. Activities of the
3,745,089
samples were then compared on the basis of light emitted
when 10 g. of ATP in solution (0.1 ml.) were added to
0.3 ml. of the sample suspension. Results are given in
the table.
RETENTION OF ACTIVITY BY FIREFLY LUCIFERASE
AFTER HEATING
Retention of activity,
percent
Compound added
Sephadex G-25^..
Biogel P-300 ..-
Dextran ---
Serum Albumin--
DEAE
Un-
heated
... 100
100
100
100
100
Heat
plus air
0
0
0
0
0
Beat
with
vacuum
10
40
0
0
0
10
15
The data shows that the firefly luciferase sterilized
under reduced pressure and in contact with Sephadex
G-25 and Biogel P-300 retained part of its activity for
reaction with ATP, while the firefly luciferase sterilized
in contact with dextran, serum albumin and DEAE lost all 20
activity.
A complete description of the preparation of luciferase
and luciferin, and their use in the method of detecting
terrestrial life, is available in the publication "The Design
and Fabrication of an Instrument for the Detection of 25
Adenosinetriphosphate (ATP)," NASA CR-411, March
1966, prepared for the National Aeronautics and Space
Administration by Hazleton Laboratories, Inc.
Although this invention has been described with ref-
erence to certain specific embodiments thereof, it should
be understood that the invention is not to be limited by
the specific embodiments except as defined in the
pended claims.
We claim:
1. A method of sterilizing firefly luciferase while pre-
venting denaturation thereof comprising heating said lu-
ciferase under a reduced pressure initially no higher than
10—* millimeters of mercury and at a temperature and
for a time sufficient to sterilize same, said luciferase being
contained in the pores of a molecular sieve selected from
the group consisting of cross-linked dextran gels having
a pore size corresponding to a protein and polyacrylamide
gels with a pore size corresponding to a protein. .
2. The method of claim 1, wherein said temperature is
within the range of 120° to 135° C.
3. The method of claim 1, wherein said temperature is
135° C.
4. The method of claim 3, wherein said time is 36
hours.
5. The method of claim 1, wherein said time is within
the range of from 6 to 36 hours.
References Cited
Science, vol. 155, Mar. 10, 1967, Chappelle et al, pp.
1287-88.
HOWARD E. SCHAIN, Primary Examiner
U.S. Cl. X.R.
195—103.5 R; 260—112 R


Protein sterilization method of firefly luciferase using reduced pressure and molecular sieves

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