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THE JOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 261, No. 3, Issue of January 25, pp. 1099-1104,1986
(Received for publication, February 11,1985)
Jeffrey R. KanofskySB and Bernard Axelrodll
From the $.Medical Service, Edward Hines, J r., 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
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Singlet Oxygen Production by Soybean Lipoxygenase Isozymes
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. 7 9 , 3871-3877) is a plausible mechanism for the observed singlet oxygen production. Lipoxygenase-3 was the most active isozyme.
Under the optimal aerobic conditions of p2H 7, 1 0 0 pg/ml lipoxygenase-3, 100 p~ linoleic acid, 100 NM 13-hydroperoxylinoleic acid, and air-saturated buffer, the yield of singlet oxygen was 12 +/- 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 lipoxygenase-3 produced comparable yields of singlet oxygen without added 13-hydroperoxylinoleic acid, since the lipoxygenase-2 served as an insitute source of hydroperoxide.
Lipoxygenase-1 was active only at low oxygen concentrations. 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.
Studies in this laboratory have been directed at the identification of biochemical sources of singlet oxygen using its characteristic infrared chemiluminescence. The development of spectrometers highly sensitive to the monomolecular emission band of singlet oxygen at 1268 nm has made possible detailed studies of singlet oxygen production in a number of peroxidase systems (1-5).
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Lipoxygenases represent another potential enzymatic source of singlet oxygen, but past studies
designed to detect singlet oxygen in lipoxygenase systems have had equivocal or negative results (6-9).
Chan’s preliminary report of singlet oxygen by soybean lipoxygenase was subsequently shown to be incorrect (9-11). A critical review of the literature concluded that singlet oxygen was not a significant product in lipoxygenase systems (12).
*This work was supported by Grant GM32974 from the National Institutes of Health, the Veterans Administration Research Service, and Grant PCM 83-16144 f rom the National Science Foundation. A preliminary report of this work was presented at the 69th Annual Meeting of the Federation of American Societies for Experimental Biology in Anaheim, CA, April 21-26, 1985 (Kanofsky, J . R., and Axelrod, B. (1985) Fed. Proc. 44, 1054).
The costs of publication of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence and reprint requests should be addressed Box 278, Hines VA Hospital, Hines, IL 60141.
(N.B. The reasons for varying results in higher and lower singlet oxygen production are explained in the discussion further below.
Breifly, studies showing low singlet oxygen production are conducted under conditions that inhibit / restrict singlet oxygen production to low / equivocal levels.
Studies showing high singlet oxygen production are conducted under conditions that optimize high singlet oxygen production.
Both sets of parameters are well-established in science and similar to all scientific studies which produce contrasting results when changes are made to specific influential parameters.)
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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-studied illustration of this point.
Christopher et al. (13, 14) were able to isolate two lipoxygenase isozymes, called lipoxygenase-2 and lipoxygenase-3, which had quite different biochemical properties from soybean lipoxygenase-1, the enzyme purified by Theorell.
Lipoxygenase-3 had high @-carotene co-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 condi tions unde r 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 to lipoxygenase-3.
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EXPERIMENTAL PROCEDURES
Chemiluminescence Spectrometer-The chemiluminescence spectrometer used for this study has been described previously (1-4). Either the integral of t h e chemiluminescence intensity over the total reaction period or the peak emission intensity is reported as appropriate.
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Quantitation of Singlet Oxygen Yields
Quantitative measurements of singlet oxygen production were made by comparing the time
integral of the chemiluminescence intensity of the system under study with a calibration curve derived from the integrated emission intensity of t h e hydrogen peroxide plus hypochlorous acid reaction.
The validity and limitations of this procedure have been discussed previously (2, 3). Calibration curves were obtained in deuterium oxide buffers with excess hypochlorous acid (1 mM) and a series of hydrogen peroxide concentrations of 100 PM or less. An individual calibration constant was determined from the slope of a least-squares regression line for each buffer used.
Turbidity Correction-For some of the conditions used, the reaction mixtures had significant turbidity. The decrease in 1268 nm emission caused by light scattering and absorption was estimated in the following manner. Solutions with various turbidities were made by adding
an appropriate amount of a 100 mM solution of linoleic acid solution in ethanol to a p2H 5, 50 mM, sodium acetate solution made with deuterium oxide.
Turbid solutions containing hydroperoxylinoleic acid or both linoleic acid and hydroperoxylinoleic acid were prepared in a similar manner. The optical density of each solution, referenced to a buffer without added fatty acid, was measured at 1268 nm using a Beckman DU-2 spectrophotometer whose long wavelength range was increased by using a lead sulfide detector. The optical density of these solutions was also measured a t 450 nm using a Perkin-Elmer
Lambda 3A spectrometer. Vycor cuvettes with a 1-cm path length were used.
The chemiluminescence from t h e hydrogen peroxide (100 p M ) plus hypochlorous acid (1 mM) reaction was t h e n measured for each solution and expressed a s a percentage of the light emission with no fatty acid.
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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 the amount of enzyme producing 1 pmollmin 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, but substantially below that seen when the enzyme activity is defined in terms of oxygen consumption (18, 19).
Enzyme concentrations were measured a t 280 nm using an absorbance of 14 for a 1% (w/v) solution of protein (18). Heat-inactivated lipoxygenase-3 was prepared by heating the enzyme solution to 90 ' C for 15 min.
Hydroperoxylinoleic acid was enzymatically synthesized from linoleic acid using lipoxygenase at 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 a t 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.
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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 a t 25 "C. Calibration curves for singlet oxygen production were obtained by injection of 1.5 ml of hydrogen peroxide in buffer into an equal volume of hypochlorous acid solution already in the spectrometer.
For the ceric ion plus 13-hydroperoxylinoleic acid 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 at 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-hydroperoxylinoleic acid. The oxygen content of the buffer was monitored with a Yellow Springs model 5331 oxygen sensor. This process was continued until the oxygen content had stabilized at the desired concentration. The buffer ( 3 ml) was then injected into the spectrometer.
The argon/oxygen flow was continued during the reaction t o maintain a constant oxygen 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.
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Statistical Analysis
All experiments were done in triplicate and are reported as the mean & S.E.
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DISCUSSION
Singlet Oxygen Production by Lipoxygenase-3
The evidence presented in this study strongly supports the production of singlet oxygen by lipoxygenase-3. Peroxy radical recombination via a Russell mechanism is a plausible explanation for the singlet oxygen production (28, 29). Peroxy radicals have been proposed as intermediates in lipoxygenase-catalyzed oxidations (30, 31).
These may result from the interaction of 13-hydroperoxylinoleic acid with lipoxygenase-3 in its reduced state. Under aerobic conditions, the reaction of oxygen with linoleic acid radicals (formed by the interaction of linoleic acid with lipoxygenase-3 in i t s oxidized state) represents a
second mechanism for the formation of peroxy radicals (30- 32).
The Russell mechanism predicts that one molecule of singlet oxygen will be produced for every two peroxy radicals (28, 29). 2 RR'CHOO' -+ RR'CHOH + RR'CO + 0, ( ' 4 ) (2) Under aerobic conditions, the maximum number of peroxy radicals that may be formed is equal to the sum of the initial concentrations of 13-hydroperoxylinoleic acid and linoleic acid.
Viewed in this manner , the lipoxygenase-3 system produced 12 & 0.4% of the theoretical yield. The decrease in singlet oxygen yield at low oxygen concentrations is consistent with the known decomposition of peroxy radicals into oxygen and alkyl radicals that occurs at low oxygen conditions (33).
The investigation of isotope scrambling by Schieberle et al. (31) provides additional support for the head to head reaction of peroxy radicals. Lipoxygenase-3 is also the most active isozyme for carotene co-oxidation. The singlet oxygen production demonstrated in this study is not sufficient to explain all of the p-carotene destruction seen in co-oxidation studies.
Oxidation of p-carotene by singlet oxygen is inefficient, since as many as 250 molecules of singlet oxygen will be quenched for each molecule of 0-carotene that is oxidized (34). The peroxy radical is more likely o be the oxidant responsible for @-carotene destruction (32, 35).
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Ceric Ion plus 13-Hydroperoxylinoleic Acid System
The ceric ion plus 13-hydroperoxylinoleic acid reaction was studied a s a simple chemical system for producing singlet oxygen via the recombination of peroxy radicals (36).
While a spectral analysis of the chemiluminescence in this system was reported to demonstrate the dimole singlet oxygen bands , in fact, the spectrum obtained was complex and most of the emission was not due to singlet oxygen (37).
The infrared emission of this system is consistent with singlet oxygen production. The
maximum yield of singlet oxygen was only 4.1 f 0.3% of thatpredicted by the Russell mechanism. The efficiency is lower than the enzymatic system, implying that the ceric ion plus 13-hydroperoxylinoleic acid reaction is not an efficient source of peroxy radicals.
An earlier study, demonstrating 15% of the predicted oxygen product, may have overestimated the singlet oxygen yield because the oxygen-sensitive, polarographic electrode used could not distinguish between ground state and excited oxygen molecules (36).
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Deuterium Isotope Effect
Deuterium oxide buffers produced only a 15 & 3-fold enhancement of singlet oxygen chemiluminescence in the lipoxygenase-3 system compared to the 30-40-fold enhancement seen in the peroxidase systems and the hydrogen peroxide plus hypochlorous acid reaction (1, 4 ).
This may have been due to an isotope effect which favored the formation of singlet oxygen in light water relative to other reactions consuming peroxy radicals and not producing singlet oxygen. Consistent with this hypothesis is the fact that the ceric ion plus 13-hydroperoxylinoleic acid reaction also ha s a low isotope effect.
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Singlet Oxygen Formation by Lipoxygenuse-1
Lipoxygen- ase-1 produced detectable concentrations of singlet oxygen only at low oxygen conditions. The co-oxidation of 0-carotene as well as certain dyes only under anaerobic conditions correlates with this observation (38-40).
The relatively low CO- oxidation activity of lipoxygenase-1 has been attributed to the formation of an enzyme-bound radical intermediate as opposed t o 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 lipoxygenase- 1 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 is suppressed. Thus, singlet oxygen production is limited to low oxygen concentrations. In con-
trast, lipoxygenase-3 produces radical intermediates independent of the oxygen concentration.
Therefore, singlet oxygen will be produced as long as the oxygen concentration is high enough to prevent the decomposition of peroxy radicals. The large increase in singlet oxygen production by lipoxygenase-1, which occurred when acetone was added to the reaction media, was probably caused by a decrease in the stability of the enzyme radical complex.
Organic solvents are known to greatly alter the kinetics of lipoxygenase-1 oxidations (42). In
contrast, the addition of acetone to the aerobic lipoxygenase-3 system (Table 11) or the ceric ion plus 13-hydroperoxylinoleic acid system (data not shown) had only a modest effect on the singlet oxygen yield.
Spectral analysis of the visible chemiluminescence that accompanies the lipoxygenase-catalyzed oxidation has failed to clearly demonstrate the dimole emission bands at 634 and 703 nm (8, 43). Boveris e t al. (43) did report a small shoulder at 630 nm, but no emission peak at 703 nm.
Unfortunately, the conditions used by these investigators did not favor the formation of singlet oxygen in our study.
Thus, in addition to the peroxidases, the lipoxygenases represent a second class of enzymes capable of producing singlet oxygen in model systems. The physiological importance of the phenomenon has yet to be determined.
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Full Article - with Results / Tables / Charts:
http://www.psi.cz/ftp/publications/singlet_oxygen/1099.pdf
|