Download Singlet Oxygen Production by Soybean Lipoxygenase Isozymes”

THEJ O U R N A L
OF
Vol. 261,No. 3, Issue of January 25,pp. 1099-1104,1986
Printed in U.S.A.
BIOLOGICAL
CHEMISTRY
Singlet Oxygen Production by Soybean Lipoxygenase Isozymes”
(Received for publication, February 11,1985)
Jeffrey R. KanofskySB and Bernard Axelrodll
From the $.Medical Service, Edward Hines, Jr., Veterans Administration Hospital, Hines, Illinois 60141 and the Loyola
University, Stritch School of Medicine, Maywood, Illinois 60153 and the TDepartment of Biochemistry, Purdue University,
West Lafayette, Indiana 47907
Lipoxygenase-catalyzed oxidations are complex processes,
however, whose intermediates may depend upon the specific
lipoxygenase used, the substrate, and the
reaction conditions.
The co-oxidation of 0-carotene by soybean lipoxygenase isozymes provides a well-studiedillustration of this point. Christopher et al. (13, 14) were able to isolate two lipoxygenase
isozymes, calledlipoxygenase-2 and lipoxygenase-3, which
had quite different biochemical properties from soybean lipoxygenase-1, the enzyme purified by Theorell. Lipoxygenase-3 hadhigh @-caroteneco-oxidation activity under aerobic
or anaerobic conditions provided a source of hydroperoxide
was present, while lipoxygenase-1 (15)and lipoxygenase-2
were relatively inactive (16, 17). The co-oxidation of 0-carotene, a well-known singlet oxygen quencher, may provide a
clue to the conditions under
which singlet oxygen is produced.
Since past attempts to demonstrate
singlet oxygen production
by lipoxygenases have used commercial preparations composed predominantly of lipoxygenase-l(6,7,9), we undertook
a study of the infrared chemiluminescence of the three soybean isozymes under a variety of reaction conditions with
particular attention tolipoxygenase-3.
EXPERIMENTALPROCEDURES
Chemiluminescence Spectrometer-The chemiluminescence spectrometer used for this study has beendescribed previously (1-4).
Studies in this laboratoryhave been directed at the identi- Either the integralof the chemiluminescence intensity over the total
fication of biochemical sources of singlet oxygen using its reaction period or the peak emission intensity is reported as appropriate.
characteristic infrared chemiluminescence. The development
Quantitation of Singlet Oxygen Yields-Quantitative measurements
of spectrometers highly sensitive to themonomolecular emis- of singlet oxygen production were made by comparingthetime
sion band of singlet oxygen at 1268 nm has made possible integral of the chemiluminescence intensity of the system under study
detailed studies of singlet oxygen production in a number of with a calibration curve derived from the integrated emission intensity of the hydrogen peroxide plus hypochlorous acid reaction. The
peroxidase systems (1-5). Lipoxygenases represent another
potential enzymaticsource of singlet oxygen, but past studies validity and limitations of this procedure have been discussed previously (2, 3). Calibration curves were obtained in deuterium oxide
designed todetectsinglet oxygen in lipoxygenase systems buffers
with excess hypochlorous acid (1mM) and a seriesof hydrogen
have had equivocal or negative results (6-9). Chan’s prelimi- peroxide concentrations of 100 PM or less. An individual calibration
nary report of singlet oxygen by soybean lipoxygenase was constant was determined from the slope of a least-squares regression
subsequently shown to be incorrect (9-11). A critical review line for each buffer used.
Turbidity Correction-For some of the conditionsused, the reaction
of the literature concluded that singlet oxygen was not a
mixtures had significant turbidity. The
decrease in 1268 nm emission
significant product in lipoxygenase systems (12).
caused by light scattering and absorption
was estimated in thefollowing manner. Solutions withvarious turbidities were made by adding
* This work was supported by Grant GM32974 from the National an appropriate amountof a 100 mM solution of linoleic acid solution
Institutes of Health, the Veterans Administration ResearchService, in ethanol to a p2H 5, 50 mM, sodium acetate solution made with
hydroperoxylinoleic
and Grant PCM 83-16144 from the National Science Foundation. A deuterium oxide. Turbidsolutionscontaining
acid or both linoleic acid and hydroperoxylinoleic acid were prepared
preliminary report of this work was presented at the 69th Annual
in a similar manner. The optical density of each solution, referenced
Meeting of the Federation of American Societies for Experimental
Biology in Anaheim, CA, April 21-26, 1985 (Kanofsky, J. R., and to a buffer without added fatty acid, was measured at 1268 nm using
Axelrod, B. (1985) Fed. Proc. 44, 1054). T h e costs of publication of a Beckman DU-2 spectrophotometer whose long wavelength range
this article were defrayed in part by the payment of page charges.
was increased by using a lead sulfide detector. The optical density of
This article must therefore
be hereby marked “aduertisernent” in these solutions was also measured at 450 nm using a Perkin-Elmer
accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.
Lambda 3A spectrometer. Vycor cuvettes with a 1-cm path length
§ T o whom correspondence and reprint requests should
be ad- were used. The chemiluminescence from the hydrogen peroxide (100
dressed Box 278, Hines VA Hospital, Hines, IL 60141.
p M ) plus hypochlorous acid (1 mM) reaction was then measured for
1099
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The oxidation of linoleic acid catalyzed by soybean
lipoxygenase isozymes was accompanied by 1268 nm
chemiluminescence characteristic of singlet oxygen.
The recombination of peroxy radicals as first proposed
by Russell (Russell, G . A. (1957) J. Am. Chem. SOC.79,
3871-3877) is a plausible mechanism for the observed
singletoxygen production. Lipoxygenase-3was the
most active isozyme. Under the optimal aerobic conditions ofp2H 7, 100 pg/ml lipoxygenase-3, 100 p~
linoleic acid, 100 NM 13-hydroperoxylinoleic acid, and
air-saturated buffer, the yield of singlet oxygen was
12 f 0.4 p~ or 12% of the amount predicted by the
Russell mechanism. High yields of singlet oxygen required the presence of 13-hydroperoxylinoleic acid.
Systems containing lipoxygenase-2 and lipoxygenase3 produced comparable yields of singlet oxygen without added13-hydroperoxylinoleic acid, sincethe lipoxygenase-2 servedas an i n situ source of hydroperoxide.
Lipoxygenase-1 was activeonly at low oxygenconcentrations. Its singlet oxygen-producing capacity was
greatly increased by the addition of acetone to the
system. Lipoxygenase-2 did not produce detectable
quantities of singlet oxygen.
1100
Singlet Oxygen Production by Soybean Lipoxygenase Isozymes
tions a factor of 30 lower than previous measurements and
the use of deuterium oxide buffers. When equimolar concentrations of reactants were used at low concentrations, the
emission was lower than that extrapolated from higher concentrations. This deviation was due to the poor stability of
very dilute hypochlorous acid solutions (24). As shown in Fig.
1, there was a linear relationship between the hydrogen peroxide concentration andthe
chemiluminescence integral
when excess hypochlorous acid was used.
Turbidity Correction-Thedesign
of the chemiluminescence spectrometer with an aluminum reflector directing the
scattered light toward the detector as well as the relatively
long wavelength of light tended to minimize the reduction in
chemiluminescence caused by turbidity. For example, the
addition of 500 PM linoleic acid and 500 PM hydroperoxylinoleic acid to p2H 5, 50 mM sodium acetate resulted in a
turbid solution, with an optical density of 0.45 at 450 nm. At
1268 nm, however, the optical density was only 0.19.
hyThe chemiluminescence of the hydrogen peroxide
pochlorous acid reaction in this quite turbid buffer was 0.96
+: 0.03 of that seen in a buffer without fatty acid. The data
presented were not corrected for turbidity, since the correction
required was usually less than the error in the measurement.
Evidence Supporting the Production of Singlet Oxygenby
Soybean Lipoxygenme-3"As shown in Figs. 2A and 6, as well
as in Tables I and 11, the oxidation of linoleic acid by lipoxygenase-3 is accompanied by chemiluminescence at 1268 nm,
characteristic of singlet oxygen. The emission has the correct
+
FIG. 1. Calibration curve for singlet oxygen production USing the hydrogen peroxide plus hypochlorous acid. Conditions
were p2H 7, 50 mM sodium phosphate, 99% deuterium oxide, 0.8%
ethanol(v/v), 1 mM hypochlorous acid. The size of the symbols
approximates the standard error.
0 30 60
RESULTS
Quuntitation of Singlet Oxygen Production-In past studies
(3), one of us (J. R. K.) has used the hydrogen peroxide plus
hypochlorous acid reaction as a calibration standard. Linear
calibration curves were obtained with equimolar concentrations of reactants in water (3). This study required an extension of the calibration procedure to singlet oxygen concentra-
I
(5)
FIG. 2. Chemiluminescence at 1268 nm from lipoxygenase
systems. A, 60 lg/ml lipoxygenase-3, p2H 7, 50 mM sodium phosphate, 200 FM linoleic acid, 200 ~ L M13-hydroperoxylinoleicacid, 99%
deuterium oxide, 0.8%ethanol (v/v), air-saturated;B, 80 pg/ml lipoxygenase-1, p2H 9, 50 mM sodium borate, 200 p M linoleic acid, 200 pM
13-hydroperoxylinoleicacid, 99% deuterium oxide, 0.8% ethanol (v/
v), 7.2% acetone (v/v), 26 p~ oxygen.
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each solution and expressed as a percentage of the light emission
with no fatty acid.
Reagents-The lipoxygenase isozymes 1-3 were isolated from soybeans and purified by previously described methods (18). These were
homogenous on disc electrophoresis (18).Lipoxygenses 1-3 had activities of 53, 34, and 4 units/mg, respectively. One unit of activity
was defined as theamount of enzyme producing 1pmollmin product
(18).Isozyme activities were measured using the ultraviolet absorption bands of characteristic products. The following conditions were
used lipoxygenase-1, pH 9, linoleic acid substrate, 234 nm band of
hydroperoxide product; lipoxygenase-2,pH 6.1, arachidonic acid substrate, 238 nm band of hydroperoxide product; lipoxygenase-3, pH
6.5, linoleic acid substrate, 280 nm band of dienone product (18). The
activity of lipoxygenase-3 using this method is comparable to that
previously reported, butsubstantially below that seen when the
enzyme activity is defined in terms of oxygen consumption (18, 19).
Enzyme concentrations were measured at 280 nmusing an absorbance
of14 for a 1% (w/v) solution of protein (18). Heat-inactivated
lipoxygenase-3was prepared by heating the enzyme solution to 90 'C
for 15 min.
Hydroperoxylinoleic acid was enzymatically synthesized from linoleic acidusing lipoxygenase a t 0 "C in the presence of excess oxygen.
About 90% of the hydroperoxide produced is the 13-hydroperoxy
isomer (20). The product had no discrete absorption band at 280 nm.
The hydroperoxide was assayed by absorbance at 234 nm using an
extinction coefficient of 2.5 X io' M" cm" (21).
Deuterium oxide (99.8%), histidine, horseradish peroxidase (Type
VI, for assay of hydrogen peroxide), linoleic acid (99%), soybean
lipoxygenase (Type I, used for synthesis of hydroperoxylinoleic acid
only), and nordihydroguaiaretic acid were obtained from Sigma. Oxygen (99.6%)was obtained from Matheson Gas Products, while argon
(99.998%)was obtained from Airco. Hypochlorous acid was distilled
from a 5.25% commercial solution (Clorox) and assayed as previously
described (3). Hydrogen peroxide (30% stabilized reagent grade, J. T.
Baker Superoxol) was assayed using the method of Cotton and
Dunford (22). All other inorganic chemicals as well as the acetone
and ethanol were reagent grade. Water was glass-distilled.
Experimental Conditions-Most experiments were done in buffers
made with deuterium oxide which enhanced the emission by a factor
of 30 (1). The apparent pH as measured with a glass electrode was
adjusted to a value 0.4 higher than the desired p2H (23). All systems
were studied at 25 "C. Calibration curves for singlet oxygen production were obtained by injection of 1.5 mlof hydrogen peroxide in
buffer into an equal volume of hypochlorous acid solution already in
the spectrometer. For the ceric ion plus 13-hydroperoxylinoleicacid
reaction, 1.5 ml of the hydroperoxide in buffer was injected into an
equal volume of buffer containing ceric ammonium nitrate. For the
lipoxygenase systems, 1.5 ml of buffer with enzyme was placed in the
spectrometer. The reaction was then initiated by the rapid injection
of an equal volume of buffer containing linoleic acid and 13-hydroperoxylinoleic acid.
For studies a t low oxygen concentrations, the lipoxygenase isozyme
in 10 p1 of buffer was placed in the spectrometer in a glass tube which
was sealed except for a small gas exit hole and flushed with an argon/
oxygen mixture of the desired proportions via a Teflon@tube. The
argon/oxygen mixture was simultaneously bubbled through buffer
containing linoleic acid and 13-hydroperoxylinoleicacid. The oxygen
content of the buffer was monitored with a Yellow Springs model
5331oxygen sensor. This process was continued until the oxygen
content had stabilized at thedesired concentration. The buffer (3 ml)
was then injected into the spectrometer. The argon/oxygen flow was
continued during the reaction to maintain a constantoxygen concentration. The gas bubbles displaced some of the buffer out of view of
the infrared detector which decreased the intensity of the signal by
32% in control experiments. This correction factor is applied to the
data presented.
Statistical Analysis-All experiments were done in triplicate and
are reported as the mean & S.E.
TABLEI
Spectral analysis of the infrared chemiluminesce~ein the
tipoxygenase-3 system, the 13-hydroperoxyli~leicacid plus ceric ion
reaction, and the hydrogen peroxide plus hypochlorous acid reaction
Chemiluminescence"
Filter
Lipoxygenase-Sb
Hydroperoxylinoleic
acid + ceric ion<
Hydrogen peroxide
+ hYPochlorous
acidd
nrn
*
*
l3-tiydroperoxylinoleic Acid (JAM)
FIG. 3. Effect of 13-hy~operoxy~ino~eic
acid concentration
on the peak intensity of the 1268 nm emission in the Iipoxygenae-3 system. Conditions were p*H7,50 mM sodium phosphate,
100pg/ml lipoxygenase-3,200 PM linoleic acid, 99% deuterium oxide,
0.8% ethanol (v/v), air-saturated. The size of the symbols approximates the standarderror.
4
5
6
TABLEIf
Effect of singlet oxygen quenchers and enzyme inhibition on 1268 nm
chemiluminescence of the lipoxygenase-3 system
Sample
Relative
chemiluminescence
Control, no additions'
1.00 f 0.03
Control + 2 mM sodium azide
0.17 f 0.03
Control + 2 mM histidine
0.39 it 0.03
Control + 0.7 mM nordihydroguaiaretic
0.03 Ift 0.02
acid
0.00 f 0.01
Heated enzyme, no additions'
Control + acetone,
1.07
7.2%
f 0.05
Qp%I?,50 mM sodium phosphate, 100 pg/mi Iipoxygenase-3, 100
p~ linoleic acid, 100 p~ 13-hydro~roxylinoleicacid, 99% deuterium
oxide, 0.8% ethanol (v/v).
* Heated to 90 "C for 15 min.
2
0
4~
0
po 200 300 400
Llpoxyqenosr-3 (nqlml)
: i rJ
1
4
0
spectraldistribution,
is inhibited by the singlet oxygen
quenchers histidine and azide ion, and is a factor of 15 f 3
more intense in 99% deuterium oxide than in light water.
Enzyme activity is required for singlet oxygen production,
since no emission was detected when the enzyme was heatinactivated. Also, nordihydroguaiaretic acid, an antioxidant
and lipoxygenase inhibitor, strongly inhibited the emission
(25).
Effect of Reaction Conditions on the P ~ o ~ ~ coft Singlet
i~n
Oxygen by S o y ~ ~u ~~~ x y g e n ~ e - 3 - F3i gshows
.
the effect
of the on cent ration of 13-hydrope~xylinoleicacid on the
reaction rate as estimated from the peak emission intensity.
With no added hydroperoxide, the onset of emission was
delayed several seconds and thepeak intensity was very low.
The half-life of the emission was prolonged, however, so that
the total singlet oxygen production was significant. Integration over a8-min period appeared to encompass all the
chemiluminescence and gave a singlet oxygen yield of 2.3 f
0.1 PM. This system must be regarded as having some hydroperoxide present, since the experimental technique exposed
the linoleic acid to air. Similar to the results of co-oxidation
studies, Iipoxygenase-8could replace the added hydroperoxide
to give comparable peak emission intensities and singlet oxygen yields (16). For example, at pZH 7, the peak emission
0
100 200
300
Linoleic k i d (#MI
400
FIG.4. Effect of reaction conditions on 1268 nm cherniluminescence of the lipoxygenase-3 system. A, p2H dependence.
Conditions were 50 mM buffers, p2H 4 and p2H 5 sodium acetate, pZH
6, pZH7, and pZH8 sodium phosphate, p2H 9 sodium borate, 100 pg/
ml lipoxygenase-3, 100 pM linoleic acid, 100 pM 13-hydroperoxylinoieic acid, 99% deuterium oxide, 0.8% ethanol (v/v), air-saturated. B,
enzyme dependence. Conditions were p2H 7, 50 mM sodium phosacid, 99%
phate, 100 PM linoleic acid, 100 p~ l3-~~droperoxylino~eic
deuterium oxide, 0.8% ethanoi (v/v), air-saturated. C, linoleic acid
dependence. Conditions were p2H 7, 50 mM sodium phosphate, 100
pg/mI lipoxygenase-3, 100 p M 13-hydroperoxylino~eicacid, 99% deuterium oxide, 0.8% ethanol (v/v), air-saturated. The size of the
symbols approximates the standarderror.
and the singlet oxygen yield of a system containing 100 bg/
ml lipoxygenase-2, 100bg/ml lipoxygenase-3, and 200 ,AM
linoleic acid were, respectively, 105 +. 18 and 97 f 5% of those
in an equivalent system containing 100 bg/ml lipoxygenase3, 100 ,AM 13-hydroperoxylinoleic acid, and 100 ptM linoleic
acid. Most subsequent experiments were done with high concentrations of hydroperoxide rather than combinations of
lipoxygenase isoenzymes.
Fig. 4 explores the effects of reaction ~ o n ~ t i o on
n s the
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0.000 f 0.002
0.00 -+ 0.04
0.00 f 0.01
0.002 0.002
0.00 0.02
0.03 f 0.03
1.00 4 0.02
1.00 -+ 0.03
1.00 f 0.02
0.59 f 0.01
0.55 f 0.03
0.48 f 0.04
0.14 f 0.02
0.16 f 0.05
0.19 f 0.04
0.034 f 0.004
0.00 f 0.03
0.01 f 0.04
-0.04 f 0.03
0.014
f 0.002
1680
0.03 4 0.07
a Each system was normalized so that the emission through the
1268-nm filter was 1.0. Emission intensities were corrected for filter
transmission and detector response.
* 80 pg/ml lipoxygenase-3, 300 p~ linoleic acid, 300 p M 13-hydroperoxylinoleic acid, 0.8% ethanol (v/v),7.2%acetone (v/v), deuterium
oxide solvent, p2H 7.0, 50 mM sodium phosphate.
500 pM 13-hydroperoxylinoleic acid, 500 p M ceric ammonium
nitrate, 20 mM hydrochloric acid, 0.8% ethanol (v/v), 7.2% acetone
(v/v), deuterium oxide solvent.
0.5 mM hydrogen peroxide, 0.5 mM hypochlorous acid, 98% deuterium oxide, pzH 7.0, 100 mM sodium phosphate, 100 mM sodium
chloride.
1070
1170
1268
1377
1475
1580
Singlet
Oxygen
Production
Soybean
byLipoxygenase
Isozymes
1102
-
hydrogenperoxide, 0.8% ethanol (v/v). 0, lipoxygenase-3system.
Conditions were p H 7, 50 mM sodium phosphate, 100 pg/ml lipoxygenase-3, 100 p~ 13-hydroperoxylinoleic acid, 100 p M linoleic acid,
0.8% ethanol (v/v), air-saturated. The standard error is shown only
when it exceeds the size of the symbols.
DISCUSSION
SingletOxygenProduction
by Lipoxygenase-3”The evidence presented in this study
strongly supports the production
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yield of singlet oxygen. The effects of p2H, linoleicacid
concentration, and enzyme concentration on singlet oxygen
yield correlate with those
previously reported for the variation
of enzyme activity with these conditions(14). Under optimal
conditions with air-saturated p2H7 buffer, 100 pg/ml lipoxygenase-3,100 p~ 13-hydroperoxylinoleicacid, and 100 p~
Time ( d
linoleic acid, the yield of singlet oxygen was 12 f 0.4 p ~ .
Fig. 5 shows the decrease in singlet oxygen yield at low
oxygen concentrations. At the lowest oxygen concentration
studied (02< 0.5 p ~ ) no
, singlet oxygen was detected.
Deuterium Isotope Effect-A shown in Fig. 6, the chemiluminescence in the lipoxygenase-3 system was enhanced by
only a factor of 15 f 3 by buffers containing 99% deuterium
oxide compared to the 30-40-fold enhancement seen in the
hydrogen peroxide plus hypochlorous acid reaction and in the
peroxidase systems (1, 4). For this reason, theemission inteCeric Ion and13-Hydroperoxylinoleic
Acid (“1
grals for both the lipoxygenase-3 system and the hydrogen
peroxide plus hypochlorous acid reaction were determined as
FIG.7. Chemiluminescence at 1268 nm for the ceric ion
a function of the deuterium oxide content of the buffer. The plus 13-hydroperoxylinoleic acid reaction. Conditions were 20
ammonium nitrate and 13reduction in chemiluminescence in light water is due both to mM hydrochloricacid,equimolarceric
the shorter half-life of singlet oxygen in light water and the hydroperoxylinoleic acid, 99% deuterium oxide, 0.8% ethanol (v/v).
time course of chemiluminescence, 500 p~ reactants; B, dependincreased optical density of light water at 1268 nm (26, 27). A,
ence of singlet oxygen yield on the concentration of reactants. The
If thelightabsorption follows Beer’s law and the singlet
size of the symbols approximates the standard error.
oxygen quenching isdescribed by the Stern-Volmer equation,
then the lightemission can be expressed as:
where x is the mole fraction of light water, Lo is the emission
seen in a deuterium oxide buffer with no light water,L, is the
L / L , = emY& + [1 - XI)
(1)
emission seen in a buffer with x amount of light water, a is a
parameter related to the absorptionof luminescence by light
water, and ,B is the ratio of the half-life of singlet oxygen in
deuterium oxide compared to light water. A nonlinear leastsquares fit of this expression to the data for the hydrogen
peroxide plus hypochlorous acid reaction is shown in Fig. 6
and gave a value of 17.5 for p, in excellent agreement with
-r 4
the value of 16.7 obtained from direct measurements of the
x 2
half-life of singlet oxygen (27).
0
Ceric Ion plus 13-Hydroperoxylinoleic Acid Reaction-The
0
50
100 150 200 250
recombination of peroxy radicals via a Russell mechanism
Oxygen (I”
represents a plausible mechanism for singlet oxygen producFIG. 5. Effect of oxygen concentration of the singlet oxygen tion in the lipoxygenase-3 system (28-31). For this reason,
production by the lipoxygenase-3 system and the lipoxygen- the infrared chemiluminescence of a simple chemical peroxy
ase-1 system. A, p2H 7, 50 mM sodiumphosphate, 200 p~ 13radical-generating system, theceric ion plus 13-hydroperoxhydroperoxylinoleic acid, 200 p~ linoleic acid,60 pg/ml lipoxygenase3,99% deuterium oxide, 0.8% ethanol (v/v). 0,p2H 9,50mM sodium ylinoleic acidreaction, was also studied(32). Fig. 7 A and
borate, 200 p~ linoleic acid, 200 p~ 13-hydroperoxylinoleic acid, 80 Table I demonstrate the characteristic infrared emission of
rg/ml lipoxygenase-1, 99% deuterium oxide, 0.8% ethanol (v/v). 0, singlet oxygen seen in this system. Fig. 7B shows the effect
lipoxygenase-1 with the same conditions above
as except 7.2% acetone of reactant concentration on singlet oxygen yield. The effi(v/v) added.
ciency of singlet oxygen production was independent of reactant concentration over the range studied. The chemiluminescence in 99% deuterium oxide was 8 k 2 times greater
than that in light
water (measured with 500 pM reactant
concentrations).
Singlet Oxygen Production by Lipoxygenme-1 and Lipoxygenme-2-As shown in Fig. 5, lipoxygenase-1 produced detectable quantitiesof singlet oxygen only at low oxygen concentrations. Its singlet
oxygen-generating activity was greatly
increased by the addition of acetone to the system. Lipoxygenase-2 did not produce detectable quantities of singlet OXygen at theconditions studied. At p2H 7 , 100 FM linoleic acid,
100 p~ 13-hydroperoxylinoleic acid, 0.1 mg/ml lipoxygenase2, the yield of singlet oxygen was 0.1 f 0.1 pM. Low oxygen
Deuterium Oxide (Mde Fraction)
,
acetone (7.2% v/v), or both low
concentration (26 p ~ )added
FIG.6. Deuterium isotope effect on 1268 nm chemiluminesoxygen
concentration
and
added
acetone failed to increase
cence. 0,hydrogen peroxide plus hypochlorous acid. Conditions were
pH 7, 50 mM sodium phosphate, 1 mM hypochlorous acid, 100 p M the 1268 nm emission.
Singlet
Oxygen
Production
Soybean
by
Lipoxygenase
Isozymes
1103
relates with this observation (38-40). The relatively low COoxidation activityof lipoxygenase-1 has been attributed to the
formation of an enzyme-bound radical intermediate as opposed to lipoxygenase-3 which may form free peroxy radicals
(32).Anaerobic conditions may lower the stability of the
enzyme-radical complex (35, 41). The oxygen concentration
requirements for singlet oxygen production by lipoxygenase1 are easily rationalized. Under strict anaerobic conditions,
the peroxy radicals would decompose before forming singlet
oxygen. At high oxygen concentrations, radical production by
lipoxygenase-1 production issuppressed. Thus, singlet oxygen
production is limited to low oxygen concentrations. In contrast, lipoxygenase-3producesradical
intermediatesindependent
of
the
oxygen
concentration.
Therefore,
singlet oxy2 RR'CHOO' -+ RR'CHOH + RR'CO + 0, ( ' 4 )
(2)
gen will be produced as long as the oxygen concentration is
Under aerobic conditions, the maximum number of peroxy high enough to prevent thedecomposition of peroxy radicals.
of the initial The large increase in singlet oxygen production by lipoxygenradicals that may be formed is equal to the sum
concentrations of 13-hydroperoxylinoleic acid and linoleic
ase-1, which occurred when acetone was added to the reaction
acid. Viewed in this manner, thelipoxygenase-3 system promedia, was probably caused by a decrease in the stability of
duced12 & 0.4%of the theoretical yield. The decrease in
the enzyme radical complex. Organic solvents are known to
singlet oxygen yield at low oxygen concentrations is consistent
greatly alter the kineticsof lipoxygenase-1 oxidations (42). In
with the known decomposition of peroxy radicals into oxygen contrast, the additionof acetone to the aerobic lipoxygenaseand alkyl radicals that occurs atlow oxygen conditions (33). 3 system (Table 11) or the ceric ion plus 13-hydroperoxylinoThe investigation of isotope scrambling by Schieberle et al.
leic acid system (data not shown) had
only a modest effect on
(31) provides additional support for the head to head reaction
the singletoxygen yield.
of peroxyradicals.Lipoxygenase-3
is also the most active
Spectralanalysis of the visible chemiluminescence that
isozyme for carotene co-oxidation. The singlet oxygen proaccompanies the lipoxygenase-catalyzed oxidation has failed
duction demonstratedin this studyis not sufficient to explain
to clearly demonstrate thedimole emission bands at 634 and
all of the p-carotene destruction seen co-oxidation
in
studies.
703 nm (8, 43). Boveris et al. (43) did report a small shoulder
Oxidation of p-carotene by singlet oxygen is inefficient, since
at 630 nm, but no emission peak at 703 nm. Unfortunately,
as many as 250 molecules of singlet oxygen will be quenched
the conditions used by these investigators did not favor the
for each molecule of 0-carotene that is oxidized (34). T h e
formation of singlet oxygen in our study.
peroxy radical is more likely to be the oxidant responsible for
Thus,inadditiontothe
peroxidases, the lipoxygenases
@-carotene destruction (32,35).
represent a second class of enzymes capable of producing
Ceric Ion plus 13-Hydroperoxylinoleic Acid System-The
singlet oxygen in model systems. The physiological imporceric ion plus 13-hydroperoxylinoleic acid reaction was studtance of the phenomenon has yet to be determined.
ied as a simple chemical system for producing singlet oxygen
via the recombinationof peroxy radicals (36). While aspectral
Acknowledgments-We wish tothank William Wardmanand
was reported Katherine Federowicz for technical assistance in performing experianalysis of the chemiluminescence in this system
to demonstrate the dimole singlet oxygen bands, in fact, the ments, Larry Kynast and Brian Dunlap for technical assistance in
spectrum obtainedwas complexand mostof the emissionwas the construction of specialized apparatus for these studies, and Shirnot due to singletoxygen (37). The infrared emission of this ley Zwiesler for assistance in preparation of the manuscript.
system is consistentwithsinglet
oxygen production.The
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1
.
J.
R.
(1983)
J . Biol. Chem. 258, 5991-5993
Kanofsky,
predicted by the Russell mechanism. The efficiency is lower
2. Kanofsky, J. R. (1984) J . Photochem. 25, 105-113
than the enzymatic system, implying that the ceric ion plus
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4061
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(1, 4). This may have been due to an isotope effect which
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to other reactions consuming
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singlet oxygen will be produced for every two peroxy radicals
(28, 29).
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