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The enzyme catalyzed oxidation of Cypridina luciferin
Vol. 31, No. 3, 1968
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE ENZYME CATALYZED OXIDATION CYPRIDINA
LUCIFERIN
Henry Program Princeton
OF
Stone
in Biochemical Sciences, Moffett Hall University, Princeton, New Jersey 08540
Received March 26, 1968 Evidence in Fig. (L-H)
has been found
1, for
the
by Cypridina
in accord
with
which
the pathway
presented
catalytic
oxidation
of Cypridina
luciferin
luciferin
oxygenase
(CLO).
mechanism is
the conclusions
chemiluminescent
supports
oxidation
LUCIFERIN
of McCapra
of a Cypridina
= L-H
This
and Chang (1967) luciferin
PEROXYLUCIFERIN
for
the
analogue.
=
L-OOH
OP” [L-OOH]
__j
•t
OXYLUCIFERIN
OXY
Hz
J-+
CO*
+
hv(460mp)
= OXY
+
CH&$OOH
R ETIOWClFERIN=
Fig.
1.
Pathway
for
CL0 in a crude of dried
organisms,
ammonium sulfate,
the
ET10
a-METHYL
degradation
BUTYRIC
ACID
of Cypridina
aqueous extract,
prepared
was precipitated
at 40 to 70% saturation
then
dialyzed,
adsorbed
386
from
luciferin. 10% by weight
on DEAE cellulose,
with
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 3 1, No. 3, 1968
eluted
by a sodium chloride
a Bio-Gel
fractions,
representing
extract,
had a specific
the crude
than
the crude
revealed
extract.
a single,
20% of the activity
slightly
unsymmetrical
reported
of
3.93
Tsuji
The stoichiometry determined with
of
by means of
of conditions Warburg
luciferin
oxidized
the Clark-Type (TABLE I).
manometers:
activity
times
Oxygen
greater material
S = 3.72. and 4.30,
Disc gel bands. to O2 consumed was Electrode
Determinations
representative
total
peak with
S values
by
The pooled
run of this
et al. (1961) reported S = 4.58. -electrophoresis showed two dark and two faint
variety
further
137
An ultracentrifuge
Shimomura et al . (1961) while
and purified
P-200 column and a second DEAE column.
most active of
gradient,
under
were also
a
made
data
are given
(TABLE II).
mpmole L-H used
*mole 02 consumed
L-H/02
TABLE I OXYGEN ELECTRODE DATA Temp oc -
M Naphos., pH 6.7 --
Act. M NaCl
Units* of CL0 x 10-6
30
0.1
0.2
0.455
55.
57.
0.97
30
0.1
0.2
0.228
110.
110.
1.00
23
0.1
0.2
0.130
270.
294.
0.92
1
0.001
0.002
0.130
100.
88.
1.10
1
0.01
0.02
0.130
100.
88.
1.10
1
0.1
0.2
0.130
100.
120.
0.84
*
The specific activity of the CL0 preparation (pooled most active fractions off the second DEAB column) used in the experiments presented in this paper, was 1.13 x lo6 activity units (arbitrary) 1 Act. Unit = 5.86 x 10B3 mpmoles of 02 per mg. protein. consumed per minute = 5.94 x log photons emitted per second.
387
Vol. 31, No. 3, 1968
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE II WARBURGDATA
pmole L-H
pmole 02 Before acid
9
3.02
2.97
1.02
---_
----
----
10 11
3.02 ----
1.61* ----
1.88 ----
1.44 0.49
2.58** ----
1.17 ----
Flask v--
Temp. 19.7OC Ratio @mole L-H)/ (pole 02)
pmole CO2 After acid
rmole CO2 corrected
Ratio @mole L-H)/ (pmole C02)
Only flask 9 had 10% KOH in the center well. Each flask contained 1.25 x LO6 activity units of CL0 in 0.05 M Na-phosphate, pH 6.8, in 0.01 M NaCl. The side-arms of flasks 9 and 10 had 1.71 mg. L-Hs2HBr in 30% aqueous methanol: the side-arm of flask 11 contained HCl equivalent to the acidity of the luciferin. "Before acid" indicates the results at the end of the period of oxygen consumption. ~2S04 was then added to the side-arms of flasks.10 and 11. "After acid" indicates the results of adding the H2SO4 to the spent reaction mixture, bringing the pH to 1.5. * uncorrected for CO2 liberation. ** CO2 liberated before acidification was 1.63 pmole.
The production experiments
with
a stoichiometry
the KOH omitted After
solutions
oxidation
in preliminary
of L-H/02
was complete
on silica
in the Warburg
and the gel
case,
had an Rf corresponding
A direct
test
solution
of Ba(OH)2
flushing
with
flasks,
extracted
butanol:glacial
gave only
as a solvent,
oxyluciferin
residues
with
H20 (4:l:l) which
= 2, as in the present
data
(TABLE II).
were dehydrated
Chromatography
called
detected
by Johnson -et al. (1962) and their analysis of data, obtained without KOH in the vessel inset,
manometric indicated
of CO2 was not
with acetic
one fluorescent
methanol. acid:
spot,
to oxyluciferin,
in each
formerly
A, Kishi for
et al. (1966). -CO2 was run by placing
the
in the center
02 gas let
well
in through
388
the
of
a saturated
the Warburg
side-arm
vent,
flask, stoppering
8lOCHEMICAL
Vol. 31, No. 3, 1968
the
flask
enzyme
and then mixing
(cup).
A blank
luciferin.ZHBr excess that
in the there
conclusions
of
electrode
(side-arm)
BaC03 was formed
plus
was no doubt
substrate
enzyme flask a positive
1)
L-H/02
2)
CO2 was formed
3)
L-H/CO2 = 1
rather
than
et al.
(1965),
evidence
together
indicates
a<-keto-$-methyl
with
that valeric
acid
on silica
gel
formed
hydrolysis
on acid
oxidation
of
luciferin
led to the
acid
as reported
by Kishi
is hydrolyzed.
of the ether
extracted
of the product is presented
butyric
organic
acid,
of the catalytic
in Fig.
2.
;
;
b)
a)
12
the to
CO2 production.
oxyluciferin
H-methyl
Chromatography
b)
for
= 1
when oxyluciferin
a)
in sufficient
experiments
is formed
1) 21 3) 4)
to
compared to the blank
test
and Warburg
the
that:
Present
Fig.
with
an amount of HCl equivalent
run.
substrate
The oxygen
the
with
was also
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2.
34
Chromatography oxyluciferin.
of organic
acid
;
formed
; on hydrolysis
of
#-keto isocaproic acid oc-keto isovaleric acid isobutyric acid 640 mpm L-H.ZHCl + 7.65 x lo6 ACT. UNITS CL0 in -01 M KP04 pH 6.8 + .02 M NaCl (ether extract of ACIDIFIED SOLUTION). Spray used was -04% Bromo Oresol green aq. soln., pH adjusted to 7 (Organic acid indicator). 2,4 dinitrophenylhydrazine, .l% soln. in 2 N HCl (d-keto acid indicator). Solvent for a and b was butanol + 1% NH3. After running chromatograms, the ammonium salt of each acid was converted the organic acid + NH39 by heating for 5 minutes at 100°C.
389
F
-
-
r
0
O-
IO
30
: c * = 40 Dz
100
Fig.
IO
3.
20 m/e
Mass spectra of of oxyluciferin Schaffer Corp.) American right:
il I
70
60
h++
isolated from the hydrolysate organic acid (lines at left of each m/e number; Morganand of &-methyl butyric acid (lines at Petroleum Institute, 1951).
60
BIOCHEMICAL
Vol. 31, No. 3, 1968
Mass spectral
analysis
pattern
which
is
similar
butyric
acid,
Fig.
synthesis
etioluciferin cellulose glacial
published
of oxyluciferin
given
the anhydride
in pyridine. acid:H20
to that
methanol.
TLC on silica
to natural
oxyluciferin
similar
to that
in Fig.
for
#-methyl
gel
conclude
spectrum
of
with
this
with
and treated
gave Rf's
saturated
and
butanol:
The spot
extract
acid on a CM-
gel with
was removed
of this
butyric
was isolated
as solvent.
in butanol The U.V.
1 is supported
of H-methyl
of oxyluciferin
of natural
We therefore
spectrum
by TLC on silica (4:l:l)
Rf similar
+ 2% NH3.
gave a fragmentation
The product
column followed
butanol
the acid
to the
from
acetic
catalyzes
of
3.
The structure by its
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
an with
similar
H20, and in
material
was exactly
oxyluciferin. that
Cypridina
luciferin
oxygenase
the reaction,
Luciferin
+ 02
-
Oxyluciferin
+ CO2 + h* hax
= 460 rnp
Acknowledqements The author is grateful to Dr. Frank Johnson and Dr. Osamu Shimomura for their support and advice throughout this work. This research was supported by Grant GM 962 from the U.S. Public Health Service, NSF Grant GB-6836 and ONR contract 4246(00). References American PetrOleUm InStitUte, Project 44, Mass Spectral Data, No. 651, Washington, D. C. (1951). Johnson, F. H., Shimomura, O., Saiga, Y., Gershman, L. C., Reynolds, G. T., and Waters, J. R., J. Cell. and Comp. Physiol., 3, 85 (1962). Kishi, Y., Goto, T., and Hirata, Y., Tetra. Letters, 3427 (1966). McCapra, F., and Chang, Y. C., Chem. comm., 1011 (1967). Shimomura, O., Johnson, F. H., and Saiga, Y., J. Cell. and COmp. Physiol., 58, 113 (1961). Tsuji, F. I., and Sowinski, R., J. Cell. and COmp. PhySiOl., 58-,
125
(1961)
.
391
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE ENZYME CATALYZED OXIDATION CYPRIDINA
LUCIFERIN
Henry Program Princeton
OF
Stone
in Biochemical Sciences, Moffett Hall University, Princeton, New Jersey 08540
Received March 26, 1968 Evidence in Fig. (L-H)
has been found
1, for
the
by Cypridina
in accord
with
which
the pathway
presented
catalytic
oxidation
of Cypridina
luciferin
luciferin
oxygenase
(CLO).
mechanism is
the conclusions
chemiluminescent
supports
oxidation
LUCIFERIN
of McCapra
of a Cypridina
= L-H
This
and Chang (1967) luciferin
PEROXYLUCIFERIN
for
the
analogue.
=
L-OOH
OP” [L-OOH]
__j
•t
OXYLUCIFERIN
OXY
Hz
J-+
CO*
+
hv(460mp)
= OXY
+
CH&$OOH
R ETIOWClFERIN=
Fig.
1.
Pathway
for
CL0 in a crude of dried
organisms,
ammonium sulfate,
the
ET10
a-METHYL
degradation
BUTYRIC
ACID
of Cypridina
aqueous extract,
prepared
was precipitated
at 40 to 70% saturation
then
dialyzed,
adsorbed
386
from
luciferin. 10% by weight
on DEAE cellulose,
with
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 3 1, No. 3, 1968
eluted
by a sodium chloride
a Bio-Gel
fractions,
representing
extract,
had a specific
the crude
than
the crude
revealed
extract.
a single,
20% of the activity
slightly
unsymmetrical
reported
of
3.93
Tsuji
The stoichiometry determined with
of
by means of
of conditions Warburg
luciferin
oxidized
the Clark-Type (TABLE I).
manometers:
activity
times
Oxygen
greater material
S = 3.72. and 4.30,
Disc gel bands. to O2 consumed was Electrode
Determinations
representative
total
peak with
S values
by
The pooled
run of this
et al. (1961) reported S = 4.58. -electrophoresis showed two dark and two faint
variety
further
137
An ultracentrifuge
Shimomura et al . (1961) while
and purified
P-200 column and a second DEAE column.
most active of
gradient,
under
were also
a
made
data
are given
(TABLE II).
mpmole L-H used
*mole 02 consumed
L-H/02
TABLE I OXYGEN ELECTRODE DATA Temp oc -
M Naphos., pH 6.7 --
Act. M NaCl
Units* of CL0 x 10-6
30
0.1
0.2
0.455
55.
57.
0.97
30
0.1
0.2
0.228
110.
110.
1.00
23
0.1
0.2
0.130
270.
294.
0.92
1
0.001
0.002
0.130
100.
88.
1.10
1
0.01
0.02
0.130
100.
88.
1.10
1
0.1
0.2
0.130
100.
120.
0.84
*
The specific activity of the CL0 preparation (pooled most active fractions off the second DEAB column) used in the experiments presented in this paper, was 1.13 x lo6 activity units (arbitrary) 1 Act. Unit = 5.86 x 10B3 mpmoles of 02 per mg. protein. consumed per minute = 5.94 x log photons emitted per second.
387
Vol. 31, No. 3, 1968
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE II WARBURGDATA
pmole L-H
pmole 02 Before acid
9
3.02
2.97
1.02
---_
----
----
10 11
3.02 ----
1.61* ----
1.88 ----
1.44 0.49
2.58** ----
1.17 ----
Flask v--
Temp. 19.7OC Ratio @mole L-H)/ (pole 02)
pmole CO2 After acid
rmole CO2 corrected
Ratio @mole L-H)/ (pmole C02)
Only flask 9 had 10% KOH in the center well. Each flask contained 1.25 x LO6 activity units of CL0 in 0.05 M Na-phosphate, pH 6.8, in 0.01 M NaCl. The side-arms of flasks 9 and 10 had 1.71 mg. L-Hs2HBr in 30% aqueous methanol: the side-arm of flask 11 contained HCl equivalent to the acidity of the luciferin. "Before acid" indicates the results at the end of the period of oxygen consumption. ~2S04 was then added to the side-arms of flasks.10 and 11. "After acid" indicates the results of adding the H2SO4 to the spent reaction mixture, bringing the pH to 1.5. * uncorrected for CO2 liberation. ** CO2 liberated before acidification was 1.63 pmole.
The production experiments
with
a stoichiometry
the KOH omitted After
solutions
oxidation
in preliminary
of L-H/02
was complete
on silica
in the Warburg
and the gel
case,
had an Rf corresponding
A direct
test
solution
of Ba(OH)2
flushing
with
flasks,
extracted
butanol:glacial
gave only
as a solvent,
oxyluciferin
residues
with
H20 (4:l:l) which
= 2, as in the present
data
(TABLE II).
were dehydrated
Chromatography
called
detected
by Johnson -et al. (1962) and their analysis of data, obtained without KOH in the vessel inset,
manometric indicated
of CO2 was not
with acetic
one fluorescent
methanol. acid:
spot,
to oxyluciferin,
in each
formerly
A, Kishi for
et al. (1966). -CO2 was run by placing
the
in the center
02 gas let
well
in through
388
the
of
a saturated
the Warburg
side-arm
vent,
flask, stoppering
8lOCHEMICAL
Vol. 31, No. 3, 1968
the
flask
enzyme
and then mixing
(cup).
A blank
luciferin.ZHBr excess that
in the there
conclusions
of
electrode
(side-arm)
BaC03 was formed
plus
was no doubt
substrate
enzyme flask a positive
1)
L-H/02
2)
CO2 was formed
3)
L-H/CO2 = 1
rather
than
et al.
(1965),
evidence
together
indicates
a<-keto-$-methyl
with
that valeric
acid
on silica
gel
formed
hydrolysis
on acid
oxidation
of
luciferin
led to the
acid
as reported
by Kishi
is hydrolyzed.
of the ether
extracted
of the product is presented
butyric
organic
acid,
of the catalytic
in Fig.
2.
;
;
b)
a)
12
the to
CO2 production.
oxyluciferin
H-methyl
Chromatography
b)
for
= 1
when oxyluciferin
a)
in sufficient
experiments
is formed
1) 21 3) 4)
to
compared to the blank
test
and Warburg
the
that:
Present
Fig.
with
an amount of HCl equivalent
run.
substrate
The oxygen
the
with
was also
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2.
34
Chromatography oxyluciferin.
of organic
acid
;
formed
; on hydrolysis
of
#-keto isocaproic acid oc-keto isovaleric acid isobutyric acid 640 mpm L-H.ZHCl + 7.65 x lo6 ACT. UNITS CL0 in -01 M KP04 pH 6.8 + .02 M NaCl (ether extract of ACIDIFIED SOLUTION). Spray used was -04% Bromo Oresol green aq. soln., pH adjusted to 7 (Organic acid indicator). 2,4 dinitrophenylhydrazine, .l% soln. in 2 N HCl (d-keto acid indicator). Solvent for a and b was butanol + 1% NH3. After running chromatograms, the ammonium salt of each acid was converted the organic acid + NH39 by heating for 5 minutes at 100°C.
389
F
-
-
r
0
O-
IO
30
: c * = 40 Dz
100
Fig.
IO
3.
20 m/e
Mass spectra of of oxyluciferin Schaffer Corp.) American right:
il I
70
60
h++
isolated from the hydrolysate organic acid (lines at left of each m/e number; Morganand of &-methyl butyric acid (lines at Petroleum Institute, 1951).
60
BIOCHEMICAL
Vol. 31, No. 3, 1968
Mass spectral
analysis
pattern
which
is
similar
butyric
acid,
Fig.
synthesis
etioluciferin cellulose glacial
published
of oxyluciferin
given
the anhydride
in pyridine. acid:H20
to that
methanol.
TLC on silica
to natural
oxyluciferin
similar
to that
in Fig.
for
#-methyl
gel
conclude
spectrum
of
with
this
with
and treated
gave Rf's
saturated
and
butanol:
The spot
extract
acid on a CM-
gel with
was removed
of this
butyric
was isolated
as solvent.
in butanol The U.V.
1 is supported
of H-methyl
of oxyluciferin
of natural
We therefore
spectrum
by TLC on silica (4:l:l)
Rf similar
+ 2% NH3.
gave a fragmentation
The product
column followed
butanol
the acid
to the
from
acetic
catalyzes
of
3.
The structure by its
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
an with
similar
H20, and in
material
was exactly
oxyluciferin. that
Cypridina
luciferin
oxygenase
the reaction,
Luciferin
+ 02
-
Oxyluciferin
+ CO2 + h* hax
= 460 rnp
Acknowledqements The author is grateful to Dr. Frank Johnson and Dr. Osamu Shimomura for their support and advice throughout this work. This research was supported by Grant GM 962 from the U.S. Public Health Service, NSF Grant GB-6836 and ONR contract 4246(00). References American PetrOleUm InStitUte, Project 44, Mass Spectral Data, No. 651, Washington, D. C. (1951). Johnson, F. H., Shimomura, O., Saiga, Y., Gershman, L. C., Reynolds, G. T., and Waters, J. R., J. Cell. and Comp. Physiol., 3, 85 (1962). Kishi, Y., Goto, T., and Hirata, Y., Tetra. Letters, 3427 (1966). McCapra, F., and Chang, Y. C., Chem. comm., 1011 (1967). Shimomura, O., Johnson, F. H., and Saiga, Y., J. Cell. and COmp. Physiol., 58, 113 (1961). Tsuji, F. I., and Sowinski, R., J. Cell. and COmp. PhySiOl., 58-,
125
(1961)
.
391