- Email: [email protected]
Effect of temperature and antioxidants upon the lipoxidase-catalyzed oxidation of sodium linoleate
Effect of Temperature and Antioxidants upon the Lipoxidase-Catalyzed Oxidation of Sodium Linoleatel A. L. Tappel,2 W. 0. Lundberg From
the Division Cnioersity
of Agricultural of Minnesota, Received
and P. D. Boyer
Biochemistry St. Paul and July
and Austin,
the Home1 Minmesota
Institute
7, 1952
Studies of the effects of temperature and antioxidants upon lipoxidase activity are important fundamentally in gaining an understanding of the nature of t’he enzyme action, and particularly in relation t)o food preservation. Holman (1) determined the effects of temperature on the activity of lipoxidase and found that the temperature for optimum activity under his assay conditions was 30°C. From limited data, Holman approximated that the Qlo was about 1.6. The literat,ure concerning the effect of antioxidants on lipoxidase activit’y is not conclusive. There have been indications that a-tocopherol has a greater retarding effect on lipoxidase oxidations than certain nonbiological antioxidants (2) and that someantioxidants have no inhibitory effect (3). The effects of a limit’ed number of antioxidants has received further attention in recent studies (1, 4, 5), but to date almost no information has appeared concerning the fate of the antioxidants in lipoxidase-catalyzed oxidations. Results are reported in this paper on the determination of the activation energy of the lipoxidase-catalyzed oxidation of linoleate over a relat,ively wide t’emperature range, on the ’ Hormel Institute publication No. 78 and Paper Ko. 2874 Scientific Journal Series, Minnesota Agricultural Experiment Station. This project has been undertaken in cooperation with the Office of Naval Research. The view or conclusions are those of the authors and are not to be construed as necessarily reflecting the views or endorsement of the Department of t,he Navy. 2 Present address: Division of Food Technology, University of California, Davis, California. 293
294
TAPPEL,
LUNDBERG
AND BOYER
evaluation of the inhibitory powers of several antioxidants, and on the fate of a-tocopherol in the lipoxidase system. EXPERIMENTAL A homogeneous substrate of sodium linoleate in aqueous solution was used following the practice of previous investigators (6). The linoleate substrate consisted of linoleic acid neutralized with sodium hydroxide and dissolved in 0.1 A; ammonium hydroxide-ammonium chloride buffer at pH 9,O or 0.1 M phosphate buffer at pH 7.0. The linoleic acid, prepared by conventional bromination-debromination procedures, contained more than 99% of 9,12-octadecadienoate, of which approximately 90% was the cis, cis form. The linoleate substrate was protected from oxidation during its preparation and storage by an atmosphere of nitrogen which had been purified by passage over heated copper. A crude lipoxidase preparation was obtained by aqueous extraction of defatted soybean meal. One gram of ground defatted s$ybeans was suspended in each 10 ml. of water. After thorough mixing the suspension was centrifuged and the supernatant liquid was frozen and stored at -18°C. until used. The manometric Warburg technique was used to follow the enzymatic oxidations in some experiments. However, for the measurement of reaction rates in the very early stages of lipoxidase-catalyzed oxidations, a sensitive and accurate spectrophotometric method was developed. In this method the oxidations were conducted in the silica cells of a Beckman model DU quartz spectrophotometer equipped with a thermostated cell compartment. Fluid for the maintenance of a constant temperature was supplied to the thermospacers from a constant-temperature bath, and any selected temperature could be maintained in the silica cells within limits of &O.l”C. In studies at lower temperatures, dry oxygen was passed through the cell compartment to prevent fogging of the cells. For each assay 0.8 ml. of 7 X 1O-3 M linoleate at the desired temperature was saturated with oxygen, introduced into a silica cell, and allowed to equilibrate to the exact temperature desired. Then 0.2 ml. of a properly diluted lipoxidase solution was added with rapid mixing, and measurements of the optical density at 2325 A. were made every 15 sec. by simultaneously balancing the photoelectric circuit and observing a stop watch. In the antioxidant studies, 0.05 ml. ethanol solution of the antioxidant was introduced with a micropipet immediately before the substrate and enzyme were mixed. This small amount of ethanol did not cause any denaturation of lipoxidase as measured by its reaction velocity. RESULTS
AND DISCUSSION
Lipoxidase Assay The direct spectrophotometric method of analysis described above proved to be a very accurate method for the determination of the initial velocities of lipoxidase-catalyzed reactions. The results of trials at 30°C. with various enzyme concentrations are shown in Fig. 1. The units of lipoxidase are defined in accordance with the assay method of Bergstrom and Holman (7). The proportion of substrate oxidized in these
LIPOXIDASE
395
ACTIVITY
runs was small and the apparent straight-line relationship between diene conjugation and time during the early part of the reaction gave a valid measure of the initial ve1ocit.y. At the higher concentrations of lipoxidase the limited supply of oxygen in the cell was soon depleted and this probably accounts for the deviation from the straight-line relationship in the later measurements. Other proposed methods for assaying lipoxidase activity (7, 8, 9) lack the advantage of establishing the initial velocity of t’he reaction by a direct
0.304
UNITS
.076
30 TIME
60 OF
90
120
150
MEASUREMENTS,
UNITS
I80
210
240
SECONDS
FIG. 1. IMect of lipoxidase concentration upon the reaction rate of linoleate oxidation as measured by the direct spectrophotometric method. Units of lipoxidase concentration according to method of Bergstriim and Holman (7). Substrate was 0.0071 M sodium linoleate buffered at pH 9.0; temperature, 30°C.
measure of the oxidation products formed during the first few minutes of the reaction. In determinations of the reaction rate by the Warburg technique, an initial lag in the manometric response was observed which lasted l-3 min. This lag was greater at lower temperatures and at lower shaker speeds. Effect of Temperature Vsing the direct spectrophotometric technique, of the lipoxidase-catalyzed reaction was measured
the initial velocit,y at various tempera-
296
TAPPEL,
LUNDBERG
AND
BOYER
tures from -6” to 30°C. Measurements below 0°C. were made with supercooled liquid solutions. When the data were plotted as logarithms of the reaction rates vs. the reciprocals of the absolute temperatures, a straight line was obtained, as shown in Fig. 2. From the slope of this line the activation energy of the lipoxidase-catalyzed oxidation of linoleate was calculated to be 4300 cal./mole.
E
.3 -
a
a
: 1
.2.I 3o” 33
I
I 3EMPERA-JWRE,’ 21 13 34
I
35 ABS.
:E”P.
I $
’
5
-3.O
36
37
lo4
2. Activation energy of the lipoxidsse-catalyzed oxidation of linoleate. Initial reaction velocities were determined by the direct spectrophotometric method. Substrate was 0.0071 M sodium linoleate buffered at pH 9.0. Lipoxidase concentration was 0.076 units. FIG.
In two experiments the Warburg manometric method was used to establish the initial oxygen absorption rates at 8” and 20°C. The results, given in Table I, yielded values for the activation energy which are essentially in agreement with the value found by the direct spectrophotometric method. Since the activation energy for this reaction is lower than those for many reactions involving other enzymes found in foods, the role of lipoxidase in food spoilage compared with that of other enzymes would
LIPOXIDASE
ACTIVITY
297
presumably be relatively more important in foods stored at low temperatures. Using the value 4300 cal./mole for the activation energy for lipoxidase, and the values 7600, 11,000, and 15,400 reported elsewhere (10) for pancreatic lipase, yeast invertase, and trypsin, the calculated relative reaction velocities at 0°C. are 45.2, 25.7, 13.2, and 5.7%, respectively, of the reaction velocities for the same enzymes at 30°C. When the lipoxidase systems at temperatures below 0°C. were permitted to freeze, the reaction rates in the solid state were less than 1% of those in the liquid state at the same temperature. The greatly deTABLE Activation
Energy
Determined
I by the Manometric
Method
Each Warburg flask contained 3 ml. substrate composed of 0.0071 21fsodium linoleate buffered at pH 9.0; 1.2 units of lipouidsse. Gas phase
Osygen
Air
Temperature “C.
Initial velocity cu. mm. Ot/min.
Average inital v&xi ty cu. mm. 02 /min.
8.1 8.1 5.1
9.75
!I. 60
20.0 20.0
13.18 12.96
13.07
8.0
11.15 10.50 10.35
10.67
11.35 15.00
14.67
8.0 8.0 20.0
20.0
Activation energy Cel./mole
9.30 9.75
41w
4100
creased rates in the solid state are probably due to the much lower rates of diffusion of the reaction components. It is therefore likely that a complete freezing of lipoxidase-containing foods by means of very low temperatures would offer good protection against lipoxidase-catalyzed oxidations. E$ects of Antioxidants
Measured by the Spectrophotometric Method
The inhibitory action of several typical phenolic antioxidants in lipoxidase systems was studied. As shown in Table II, all the antioxidants tested were inhibitory, but in different degrees.Benzoquinone and several organic hydroxyl compounds, including lauryl alcohol, cholesterol, and
298
TAPPEL,
LUNDBERG
AND
BOYER
phenol, were also tested but were found to have no inhibitory effects. These observations suggest that the inhibitory action of the phenolic antioxidants on lipoxidase-catalyzed oxidations is in some way related to their inhibitory action on the autoxidation of unsaturated fatty acid compounds. Because of the important biological role of the antioxidant a-tocopherol, its inhibitory action on lipoxidase catalysis was investigated quite extensively. In one study, the results of which are summarized in Table III, the inhibitory action of a-tocopherol at 0” and 30°C. was compared with that of hydroquinone. The two antioxidants were about equally inhibitory at 30°C. At 0°C. both antioxidants were less effective, TABLE II Lipoxidase-Catalyzed Oxidation of Linoleate by Various Antioxidants Initial reaction rate was determined by the direct spectrophotometric method. Substrate was 0.0071 M sodium linoleate buffered at pH 9. Temperature, 30°C. Inhibition
of
Antioxidant
a-Naphthol a-Naphthol Catechol Catechol a-Tocopherol Hydroquinone Phloroglucinol
Concentration x lo-’ Y
1.7 17 1.7 17 1.7 1.7 1.7
Inhibition of the initial reaction rate %
41.5 100.0 49.5 97.9 36.9 26.7 13.4
and at various concentrations tested, below 2 X 1O-3M, hydroquinone, unlike cr-tocopherol, was completely ineffective at 0°C. Two commercially important antioxidants which have about the same strong inhibitory effect on the autoxidation of fatty acid esters were also tested in the lipoxidase system, and the results are included in Table III. Nordihydroguaiaretic acid (NDGA) was generally more effective than propyl gallate and both were considerably more inhibitory than a-tocopherol or hydroquinone. At 22°C. the rate of linoleate oxidation was found to be nil within the limits of experimental error, using either NDGA or propyl gallate at a concentration of 1 X 10m4M. Again, however, the inhibitory effects were less pronounced at 0°C. In another study for which no tables or figures are included, the effects of hydroquinone and NDGA on the lipoxidase-catalyzed oxidation of linoleate were compared with their effects on the autoxidation of the same system in the absence of lipoxidase at 22°C. It was found for each
LIPOXIDASE
antioxidant to produce oxidations. similarity However,
ACTIVITY
that’ approximately the same concentration was required a measurable inhibition in both the enzymic and nonenzymic Superficially this might be taken as an indication of a marked in the mechanisms of the enzymic and nonenzymic oxidations. a det’ailed analysis of the kinetics of t’he lipoxidase-catalyzed TABLE
Eject
of Antioxidant
Initial method.
299
reaction Substrate
Concentrations Reaction
III and Reaction Velocities
velocities were determined by was 0.0071 M sodium linoleate I
Antioxidant
Temperatures
Inhibition
of initial
reaction
lo-’
-
2 5 10 20
0 0 0 53.4
1 2 8 16
%
90-100 ~-
-I-
27.0 50.0 62.6 -
13.0 38.3 38.3 0°C.
22°C. -
SIX;:\
--
0.01
-
0.1
8.0
1
66.0
~-. 0
38.0 100.0 -1. -___-.
Propyl
~~
38.0 58.7 70.0 .__
!
at:
30°C.
I
%
1
-p---_~ u-Tocopherol
df
velocity
-____-..
0°C. x
Initial
the direct spectrophotometric buffered at pH 9.0.
Concentration
Hydroquinone
upon
gallate
0.0 13.0 100.0
oxidation of linoleate (11) has indicated that the mechanism of the lipoxidase reaction is probably quite different from t#hat proposed by Bolland (12) for t,he aut,oxidation of et,hyl linoleate. Sntioxidant
Studies by Oxygen Absorpiim
Further investigat’ions showed that below certain concentrations the nnti0xidant.s underwent oxidat,ion bccausc of t,he presence of lipoxidase.
300
TAPPEL,
LUNDBERG
AND
BOYER
Oxygen absorption measurements by the Warburg manometric method were used to follow the combined oxidation of linoleate and antioxidant. Table IV shows the effects of relatively high concentrations of the various antioxidants on the initial rate of oxygen absorption. It was found that antioxidants such as cY-naphthol, propyl gallate, and NDGA, which at higher concentrations were shown by the direct spectrophotometric method to decrease the linoleate oxidation rate to zero, likewise produced a marked reduction in oxygen absorption. Pyrogallol, phloroglucinol, and hydroquinone showed lesser reductions of oxygen absorption even though they caused a marked inhibition of the linoleate TABLE Inhibition Each linoleate, alcohol;
Warburg buffered 1.2 units
of Oxygen flask contained with phosphate of lipoxidase.
Antioxidant
Absorption
IV by Various
3 ml. substrate buffer at pH
Concentration
x lo-~ Y None a-Naphthol Pyrogallol Phloroglucinol Hydroquinone Propyl gallate NDGA cu-Tocopherol
Antioxidants
composed 7. Antioxidant
of 0.0036 211 sodium in 0.1 ml. ethyl Inhibition of initial reaction rate as measured by oxygen uptake, WC.
% 0 94.0 19.0 4.0 6.0 73.0 100.0 39.0
oxidation. These observations can be best explained by assuming that the antioxidants can react with the lipoxidase indirectly, i.e., that the antioxidants may be catalytically oxidized by the combina.tion of lipoxidase and linoleate. The case of lipoxidase inhibition by NDGA has been studied in detail and was found to be reversible. A mechanism for lipoxidase catalysis which offers an explanation for the oxidation of antioxidants without net oxidation of linoleate has been .postulated (11). In the case of a-naphthol, propyl gallate, and NDGA at relatively high concentrations, inhibition of lipoxidase is apparently the dominating reaction. The high oxygen absorption together with low linoleate oxidation at relatively high concentrations of pyrogallol, phloroglucinol, and hydroquinone indicates that in the case of these antioxidants, catalytic oxidation by the lipoxidase-linoleate complex is the dominating reaction.
LIPOXIDASE
ACTIVITY
302
Figure 3 shows t,he course of oxygen uptake measureme& in the lipoxidase-catalyzed oxidation of linoleate in the presence of various concentrations of cY-tocopherol at 30°C. Although there was a significant reduction in the init.ial rate of oxygen upt,ake, particularly at the highest I
I
50 TIME
OF
REACTION,
60
MINUTES
FIG. 3. The ett’rct of tocopherol on oxygen absorption by the lipoxidnse-linole;cte system. a-Tocopherol : 1, SOIIC; d, 1 X lo-” U; 3, 2 X IO+ df; 4, 2 X 10-J N; 5, 4 X lo-” .I/; h’, 4 X 10eL -II, and no enzyme. A blank run containing only linoleate substrate and no tocopherol and no liposidase did not show any oxygen absorption during the course of the csperiment. Substrate was 0.0046 Jf sodium linoleate buffered at pH 9.0 and contained 1% Tween 40.5 I&h Warburg flask contained 1.58 units lipoxidase in side arm and had an atmosphere of oxygen gas. Temperature of reaction was 30°C. concentration of a-tocopherol, in general, the reduction was not, as great a+s that found for the linoleate oxidation as measured by the direct spectrophotometric method. In this case also, therefore, it is probable that the dominating reaction taking place is an oxidation of a-t’ocopherol catalyzed by the lipoxidase-linoleate complex. a I’olyoxvalkvlene I _
derivative
of sorbitan
monopalmitate
302
TAPPEL,
LUNDBERG
AND
BOYER
The curve in Fig. 3 obtained with a relatively high concentration of cr-tocopherol in the absence of lipoxidase demonstrates a prooxidant effect of the cu-tocopherol itself, an effect that has been observed also in the autoxidation of fatty acid esters and one that is greater at higher antioxidant concentrations. It is probable that the higher levels of oxygen uptake shown by the curves for the higher antioxidant concentrations
4s IOOI
I
20
30
I
IO
TIME
The disappearance
OF
I
40
I
I
I
I
50
60
70
80
REAC-TION,
of added a-tocopherol
MINUTES
in the lipoxidase-linoleate
The system contained 30 ml. of 0.0071 M sodium linoleate buffered at pH 9.0 and 8.1 units of lipoxidase. The initial tocopherol concentration was 6 X l-0-l M. Reaction temperature was 30°C. All extracted reducing substances were calculated as a-tocopherol.
in the later stages of the oxidations are attributable to this prooxidant effect of the antioxidant. Nordihydroguaiaretic acid and propyl gallate are two antioxidant5 which might prove useful in protecting foods which undergo deteriora. tion because of unsaturated fat oxidation catalyzed by lipoxidase. These two antioxidants are generally acceptable for use in foods and are effec tive against lipoxidase catalysis at a concentration which could b maintained in edible products.
LIPOXIDASE
Oxidation
303
ACTIVITY
of a-Tocopherol
In an effort to determine the fate of a-tocopherol in the lipoxidase syst’em at 3O”C., oxidations were conducted with an initial a-tocopherol concent8ration of 6 X 1O-4 M, and petroleum ether extracts of the residual a-tocopherol and its extractable oxidation products were obtained from t’he reaction mixtures after various reaction times. The total
I ,
‘0 WAVE FIN. 5. Spectral t,he lipoxidase-linoleate
absorption changes system. Same
LENGTH,
MJJ
during the cobsidation conditions of reaction
of a-tocopherol as in Fig. 4.
iu
reducing power of the extract was determined by reaction with a ferric chloride-2,2’-bipyridine reagent (13) _ The reducing substances calculated as a-tocopherol were found to decrease greatly during the first 15 min. and more slowly thereafter, as shown in Fig. 4. The absorption spectra of extracts obtained in 0-, lo-, and 80-min. reaction times are shown in Fig. 5; the destruction of a-tocopherol is indicated by the marked decreasesin the absorption maximum in the region of 290-295
304
TAPPEL,
LUNDBERG
AND
BOYER
rnp. However, the magnitude of the increases in absorption in the region 260-270 rnp, where cr-tocopherylquinone has a high absorption maximum, indicates that at most only a small portion of the a-tocopherol was converted to the quinone. The products of the oxidation of ac-tocopherol in this system are probably mixtures of cu-tocopheroxide and reducing and nonreducing dimers which have been found to be present in the oxidation products of a-tocopherol in other systems (14). SUMMARY
The effects of temperature from -6’ to 30°C. and antioxidants upon the lipoxidase-catalyzed oxidation of aqueous sodium linoleate have been studied. An accurate analytical method for establishing the initial rates of the lipoxidase reaction has been described. The lipoxidase-catalyzed oxidation of linoleate was found to have a relatively low activation energy, 4300 cal./mole. Complete freezing of the lipoxidase system reduced the reaction rate to less than 1% of that found in the liquid system at the same temperature. All the phenolic antioxidants tested were found to inhibit the reaction. The greatest inhibitory effect was obtained with nordihydroguaiaretic acid, followed closely by propyl gallate; the effective concentrations were sufficiently low so that these two antioxidants might prove useful in protecting foods from this t,ype of oxidation. The biologically important antioxidant, cu-tocopherol, was rapidly oxidized in the lipoxidase system. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 11.
HOLMAN, R. T., ;irch. Biochem. 16, 403 (1947). GY~RC~Y, I?., AND TOMARELLI, R. RI., .T. Biol. Chem. 164, 317 (1944). STRAIN, H. H., J. Am. Chem. Sot. 63, 3542 (1941). KUNKEL, H. O., Arch. Biochem. 30, 306 (1951). HOLMAN, It. T., Trans. Third Conf. Riol. Antios., 1’. 131. Josiah Macy Jr. Foundation, New York 1948. THEORELL, H., BERGSTROM, S., ASI) .&c~:sox, A., Phnrm. .4&a Helv. 21, 318 (1946). BERGSTR~X, S., AKD HOLMAK, It. T., .~lricunces in E’ntpol. 8, 425 (1948). COSBY, E., AND SUMNER, J. B., rlrch. Biochem. 8, 259 (1945). SUMNER, J. B., AND SMITH, G. N., Arch. Biochem. 14, 87 (1947). SIZER, I. W., ANI! JOSEPHSOS, 13. S., Food Research 7, 201 (1942). TAPPEL, A. I,., BOYER, 1’. D., ASD I,I.NI)RER(:, W. 0.. .I. Biol. Chem. 199, 267 (1952). BOLLASD, J. I,., Proc. IZou. Sot. (London) A186, 218 (1946). KAUNITZ, H.. MD BEAVER. J. J., .J. Hiol. (“hem. 166, 6.53 (1911). Ii%OYER,
1’. D.,
.J. .tn~.
C’hcm.
,%c.
73.
734
(19511.
the Division Cnioersity
of Agricultural of Minnesota, Received
and P. D. Boyer
Biochemistry St. Paul and July
and Austin,
the Home1 Minmesota
Institute
7, 1952
Studies of the effects of temperature and antioxidants upon lipoxidase activity are important fundamentally in gaining an understanding of the nature of t’he enzyme action, and particularly in relation t)o food preservation. Holman (1) determined the effects of temperature on the activity of lipoxidase and found that the temperature for optimum activity under his assay conditions was 30°C. From limited data, Holman approximated that the Qlo was about 1.6. The literat,ure concerning the effect of antioxidants on lipoxidase activit’y is not conclusive. There have been indications that a-tocopherol has a greater retarding effect on lipoxidase oxidations than certain nonbiological antioxidants (2) and that someantioxidants have no inhibitory effect (3). The effects of a limit’ed number of antioxidants has received further attention in recent studies (1, 4, 5), but to date almost no information has appeared concerning the fate of the antioxidants in lipoxidase-catalyzed oxidations. Results are reported in this paper on the determination of the activation energy of the lipoxidase-catalyzed oxidation of linoleate over a relat,ively wide t’emperature range, on the ’ Hormel Institute publication No. 78 and Paper Ko. 2874 Scientific Journal Series, Minnesota Agricultural Experiment Station. This project has been undertaken in cooperation with the Office of Naval Research. The view or conclusions are those of the authors and are not to be construed as necessarily reflecting the views or endorsement of the Department of t,he Navy. 2 Present address: Division of Food Technology, University of California, Davis, California. 293
294
TAPPEL,
LUNDBERG
AND BOYER
evaluation of the inhibitory powers of several antioxidants, and on the fate of a-tocopherol in the lipoxidase system. EXPERIMENTAL A homogeneous substrate of sodium linoleate in aqueous solution was used following the practice of previous investigators (6). The linoleate substrate consisted of linoleic acid neutralized with sodium hydroxide and dissolved in 0.1 A; ammonium hydroxide-ammonium chloride buffer at pH 9,O or 0.1 M phosphate buffer at pH 7.0. The linoleic acid, prepared by conventional bromination-debromination procedures, contained more than 99% of 9,12-octadecadienoate, of which approximately 90% was the cis, cis form. The linoleate substrate was protected from oxidation during its preparation and storage by an atmosphere of nitrogen which had been purified by passage over heated copper. A crude lipoxidase preparation was obtained by aqueous extraction of defatted soybean meal. One gram of ground defatted s$ybeans was suspended in each 10 ml. of water. After thorough mixing the suspension was centrifuged and the supernatant liquid was frozen and stored at -18°C. until used. The manometric Warburg technique was used to follow the enzymatic oxidations in some experiments. However, for the measurement of reaction rates in the very early stages of lipoxidase-catalyzed oxidations, a sensitive and accurate spectrophotometric method was developed. In this method the oxidations were conducted in the silica cells of a Beckman model DU quartz spectrophotometer equipped with a thermostated cell compartment. Fluid for the maintenance of a constant temperature was supplied to the thermospacers from a constant-temperature bath, and any selected temperature could be maintained in the silica cells within limits of &O.l”C. In studies at lower temperatures, dry oxygen was passed through the cell compartment to prevent fogging of the cells. For each assay 0.8 ml. of 7 X 1O-3 M linoleate at the desired temperature was saturated with oxygen, introduced into a silica cell, and allowed to equilibrate to the exact temperature desired. Then 0.2 ml. of a properly diluted lipoxidase solution was added with rapid mixing, and measurements of the optical density at 2325 A. were made every 15 sec. by simultaneously balancing the photoelectric circuit and observing a stop watch. In the antioxidant studies, 0.05 ml. ethanol solution of the antioxidant was introduced with a micropipet immediately before the substrate and enzyme were mixed. This small amount of ethanol did not cause any denaturation of lipoxidase as measured by its reaction velocity. RESULTS
AND DISCUSSION
Lipoxidase Assay The direct spectrophotometric method of analysis described above proved to be a very accurate method for the determination of the initial velocities of lipoxidase-catalyzed reactions. The results of trials at 30°C. with various enzyme concentrations are shown in Fig. 1. The units of lipoxidase are defined in accordance with the assay method of Bergstrom and Holman (7). The proportion of substrate oxidized in these
LIPOXIDASE
395
ACTIVITY
runs was small and the apparent straight-line relationship between diene conjugation and time during the early part of the reaction gave a valid measure of the initial ve1ocit.y. At the higher concentrations of lipoxidase the limited supply of oxygen in the cell was soon depleted and this probably accounts for the deviation from the straight-line relationship in the later measurements. Other proposed methods for assaying lipoxidase activity (7, 8, 9) lack the advantage of establishing the initial velocity of t’he reaction by a direct
0.304
UNITS
.076
30 TIME
60 OF
90
120
150
MEASUREMENTS,
UNITS
I80
210
240
SECONDS
FIG. 1. IMect of lipoxidase concentration upon the reaction rate of linoleate oxidation as measured by the direct spectrophotometric method. Units of lipoxidase concentration according to method of Bergstriim and Holman (7). Substrate was 0.0071 M sodium linoleate buffered at pH 9.0; temperature, 30°C.
measure of the oxidation products formed during the first few minutes of the reaction. In determinations of the reaction rate by the Warburg technique, an initial lag in the manometric response was observed which lasted l-3 min. This lag was greater at lower temperatures and at lower shaker speeds. Effect of Temperature Vsing the direct spectrophotometric technique, of the lipoxidase-catalyzed reaction was measured
the initial velocit,y at various tempera-
296
TAPPEL,
LUNDBERG
AND
BOYER
tures from -6” to 30°C. Measurements below 0°C. were made with supercooled liquid solutions. When the data were plotted as logarithms of the reaction rates vs. the reciprocals of the absolute temperatures, a straight line was obtained, as shown in Fig. 2. From the slope of this line the activation energy of the lipoxidase-catalyzed oxidation of linoleate was calculated to be 4300 cal./mole.
E
.3 -
a
a
: 1
.2.I 3o” 33
I
I 3EMPERA-JWRE,’ 21 13 34
I
35 ABS.
:E”P.
I $
’
5
-3.O
36
37
lo4
2. Activation energy of the lipoxidsse-catalyzed oxidation of linoleate. Initial reaction velocities were determined by the direct spectrophotometric method. Substrate was 0.0071 M sodium linoleate buffered at pH 9.0. Lipoxidase concentration was 0.076 units. FIG.
In two experiments the Warburg manometric method was used to establish the initial oxygen absorption rates at 8” and 20°C. The results, given in Table I, yielded values for the activation energy which are essentially in agreement with the value found by the direct spectrophotometric method. Since the activation energy for this reaction is lower than those for many reactions involving other enzymes found in foods, the role of lipoxidase in food spoilage compared with that of other enzymes would
LIPOXIDASE
ACTIVITY
297
presumably be relatively more important in foods stored at low temperatures. Using the value 4300 cal./mole for the activation energy for lipoxidase, and the values 7600, 11,000, and 15,400 reported elsewhere (10) for pancreatic lipase, yeast invertase, and trypsin, the calculated relative reaction velocities at 0°C. are 45.2, 25.7, 13.2, and 5.7%, respectively, of the reaction velocities for the same enzymes at 30°C. When the lipoxidase systems at temperatures below 0°C. were permitted to freeze, the reaction rates in the solid state were less than 1% of those in the liquid state at the same temperature. The greatly deTABLE Activation
Energy
Determined
I by the Manometric
Method
Each Warburg flask contained 3 ml. substrate composed of 0.0071 21fsodium linoleate buffered at pH 9.0; 1.2 units of lipouidsse. Gas phase
Osygen
Air
Temperature “C.
Initial velocity cu. mm. Ot/min.
Average inital v&xi ty cu. mm. 02 /min.
8.1 8.1 5.1
9.75
!I. 60
20.0 20.0
13.18 12.96
13.07
8.0
11.15 10.50 10.35
10.67
11.35 15.00
14.67
8.0 8.0 20.0
20.0
Activation energy Cel./mole
9.30 9.75
41w
4100
creased rates in the solid state are probably due to the much lower rates of diffusion of the reaction components. It is therefore likely that a complete freezing of lipoxidase-containing foods by means of very low temperatures would offer good protection against lipoxidase-catalyzed oxidations. E$ects of Antioxidants
Measured by the Spectrophotometric Method
The inhibitory action of several typical phenolic antioxidants in lipoxidase systems was studied. As shown in Table II, all the antioxidants tested were inhibitory, but in different degrees.Benzoquinone and several organic hydroxyl compounds, including lauryl alcohol, cholesterol, and
298
TAPPEL,
LUNDBERG
AND
BOYER
phenol, were also tested but were found to have no inhibitory effects. These observations suggest that the inhibitory action of the phenolic antioxidants on lipoxidase-catalyzed oxidations is in some way related to their inhibitory action on the autoxidation of unsaturated fatty acid compounds. Because of the important biological role of the antioxidant a-tocopherol, its inhibitory action on lipoxidase catalysis was investigated quite extensively. In one study, the results of which are summarized in Table III, the inhibitory action of a-tocopherol at 0” and 30°C. was compared with that of hydroquinone. The two antioxidants were about equally inhibitory at 30°C. At 0°C. both antioxidants were less effective, TABLE II Lipoxidase-Catalyzed Oxidation of Linoleate by Various Antioxidants Initial reaction rate was determined by the direct spectrophotometric method. Substrate was 0.0071 M sodium linoleate buffered at pH 9. Temperature, 30°C. Inhibition
of
Antioxidant
a-Naphthol a-Naphthol Catechol Catechol a-Tocopherol Hydroquinone Phloroglucinol
Concentration x lo-’ Y
1.7 17 1.7 17 1.7 1.7 1.7
Inhibition of the initial reaction rate %
41.5 100.0 49.5 97.9 36.9 26.7 13.4
and at various concentrations tested, below 2 X 1O-3M, hydroquinone, unlike cr-tocopherol, was completely ineffective at 0°C. Two commercially important antioxidants which have about the same strong inhibitory effect on the autoxidation of fatty acid esters were also tested in the lipoxidase system, and the results are included in Table III. Nordihydroguaiaretic acid (NDGA) was generally more effective than propyl gallate and both were considerably more inhibitory than a-tocopherol or hydroquinone. At 22°C. the rate of linoleate oxidation was found to be nil within the limits of experimental error, using either NDGA or propyl gallate at a concentration of 1 X 10m4M. Again, however, the inhibitory effects were less pronounced at 0°C. In another study for which no tables or figures are included, the effects of hydroquinone and NDGA on the lipoxidase-catalyzed oxidation of linoleate were compared with their effects on the autoxidation of the same system in the absence of lipoxidase at 22°C. It was found for each
LIPOXIDASE
antioxidant to produce oxidations. similarity However,
ACTIVITY
that’ approximately the same concentration was required a measurable inhibition in both the enzymic and nonenzymic Superficially this might be taken as an indication of a marked in the mechanisms of the enzymic and nonenzymic oxidations. a det’ailed analysis of the kinetics of t’he lipoxidase-catalyzed TABLE
Eject
of Antioxidant
Initial method.
299
reaction Substrate
Concentrations Reaction
III and Reaction Velocities
velocities were determined by was 0.0071 M sodium linoleate I
Antioxidant
Temperatures
Inhibition
of initial
reaction
lo-’
-
2 5 10 20
0 0 0 53.4
1 2 8 16
%
90-100 ~-
-I-
27.0 50.0 62.6 -
13.0 38.3 38.3 0°C.
22°C. -
SIX;:\
--
0.01
-
0.1
8.0
1
66.0
~-. 0
38.0 100.0 -1. -___-.
Propyl
~~
38.0 58.7 70.0 .__
!
at:
30°C.
I
%
1
-p---_~ u-Tocopherol
df
velocity
-____-..
0°C. x
Initial
the direct spectrophotometric buffered at pH 9.0.
Concentration
Hydroquinone
upon
gallate
0.0 13.0 100.0
oxidation of linoleate (11) has indicated that the mechanism of the lipoxidase reaction is probably quite different from t#hat proposed by Bolland (12) for t,he aut,oxidation of et,hyl linoleate. Sntioxidant
Studies by Oxygen Absorpiim
Further investigat’ions showed that below certain concentrations the nnti0xidant.s underwent oxidat,ion bccausc of t,he presence of lipoxidase.
300
TAPPEL,
LUNDBERG
AND
BOYER
Oxygen absorption measurements by the Warburg manometric method were used to follow the combined oxidation of linoleate and antioxidant. Table IV shows the effects of relatively high concentrations of the various antioxidants on the initial rate of oxygen absorption. It was found that antioxidants such as cY-naphthol, propyl gallate, and NDGA, which at higher concentrations were shown by the direct spectrophotometric method to decrease the linoleate oxidation rate to zero, likewise produced a marked reduction in oxygen absorption. Pyrogallol, phloroglucinol, and hydroquinone showed lesser reductions of oxygen absorption even though they caused a marked inhibition of the linoleate TABLE Inhibition Each linoleate, alcohol;
Warburg buffered 1.2 units
of Oxygen flask contained with phosphate of lipoxidase.
Antioxidant
Absorption
IV by Various
3 ml. substrate buffer at pH
Concentration
x lo-~ Y None a-Naphthol Pyrogallol Phloroglucinol Hydroquinone Propyl gallate NDGA cu-Tocopherol
Antioxidants
composed 7. Antioxidant
of 0.0036 211 sodium in 0.1 ml. ethyl Inhibition of initial reaction rate as measured by oxygen uptake, WC.
% 0 94.0 19.0 4.0 6.0 73.0 100.0 39.0
oxidation. These observations can be best explained by assuming that the antioxidants can react with the lipoxidase indirectly, i.e., that the antioxidants may be catalytically oxidized by the combina.tion of lipoxidase and linoleate. The case of lipoxidase inhibition by NDGA has been studied in detail and was found to be reversible. A mechanism for lipoxidase catalysis which offers an explanation for the oxidation of antioxidants without net oxidation of linoleate has been .postulated (11). In the case of a-naphthol, propyl gallate, and NDGA at relatively high concentrations, inhibition of lipoxidase is apparently the dominating reaction. The high oxygen absorption together with low linoleate oxidation at relatively high concentrations of pyrogallol, phloroglucinol, and hydroquinone indicates that in the case of these antioxidants, catalytic oxidation by the lipoxidase-linoleate complex is the dominating reaction.
LIPOXIDASE
ACTIVITY
302
Figure 3 shows t,he course of oxygen uptake measureme& in the lipoxidase-catalyzed oxidation of linoleate in the presence of various concentrations of cY-tocopherol at 30°C. Although there was a significant reduction in the init.ial rate of oxygen upt,ake, particularly at the highest I
I
50 TIME
OF
REACTION,
60
MINUTES
FIG. 3. The ett’rct of tocopherol on oxygen absorption by the lipoxidnse-linole;cte system. a-Tocopherol : 1, SOIIC; d, 1 X lo-” U; 3, 2 X IO+ df; 4, 2 X 10-J N; 5, 4 X lo-” .I/; h’, 4 X 10eL -II, and no enzyme. A blank run containing only linoleate substrate and no tocopherol and no liposidase did not show any oxygen absorption during the course of the csperiment. Substrate was 0.0046 Jf sodium linoleate buffered at pH 9.0 and contained 1% Tween 40.5 I&h Warburg flask contained 1.58 units lipoxidase in side arm and had an atmosphere of oxygen gas. Temperature of reaction was 30°C. concentration of a-tocopherol, in general, the reduction was not, as great a+s that found for the linoleate oxidation as measured by the direct spectrophotometric method. In this case also, therefore, it is probable that the dominating reaction taking place is an oxidation of a-t’ocopherol catalyzed by the lipoxidase-linoleate complex. a I’olyoxvalkvlene I _
derivative
of sorbitan
monopalmitate
302
TAPPEL,
LUNDBERG
AND
BOYER
The curve in Fig. 3 obtained with a relatively high concentration of cr-tocopherol in the absence of lipoxidase demonstrates a prooxidant effect of the cu-tocopherol itself, an effect that has been observed also in the autoxidation of fatty acid esters and one that is greater at higher antioxidant concentrations. It is probable that the higher levels of oxygen uptake shown by the curves for the higher antioxidant concentrations
4s IOOI
I
20
30
I
IO
TIME
The disappearance
OF
I
40
I
I
I
I
50
60
70
80
REAC-TION,
of added a-tocopherol
MINUTES
in the lipoxidase-linoleate
The system contained 30 ml. of 0.0071 M sodium linoleate buffered at pH 9.0 and 8.1 units of lipoxidase. The initial tocopherol concentration was 6 X l-0-l M. Reaction temperature was 30°C. All extracted reducing substances were calculated as a-tocopherol.
in the later stages of the oxidations are attributable to this prooxidant effect of the antioxidant. Nordihydroguaiaretic acid and propyl gallate are two antioxidant5 which might prove useful in protecting foods which undergo deteriora. tion because of unsaturated fat oxidation catalyzed by lipoxidase. These two antioxidants are generally acceptable for use in foods and are effec tive against lipoxidase catalysis at a concentration which could b maintained in edible products.
LIPOXIDASE
Oxidation
303
ACTIVITY
of a-Tocopherol
In an effort to determine the fate of a-tocopherol in the lipoxidase syst’em at 3O”C., oxidations were conducted with an initial a-tocopherol concent8ration of 6 X 1O-4 M, and petroleum ether extracts of the residual a-tocopherol and its extractable oxidation products were obtained from t’he reaction mixtures after various reaction times. The total
I ,
‘0 WAVE FIN. 5. Spectral t,he lipoxidase-linoleate
absorption changes system. Same
LENGTH,
MJJ
during the cobsidation conditions of reaction
of a-tocopherol as in Fig. 4.
iu
reducing power of the extract was determined by reaction with a ferric chloride-2,2’-bipyridine reagent (13) _ The reducing substances calculated as a-tocopherol were found to decrease greatly during the first 15 min. and more slowly thereafter, as shown in Fig. 4. The absorption spectra of extracts obtained in 0-, lo-, and 80-min. reaction times are shown in Fig. 5; the destruction of a-tocopherol is indicated by the marked decreasesin the absorption maximum in the region of 290-295
304
TAPPEL,
LUNDBERG
AND
BOYER
rnp. However, the magnitude of the increases in absorption in the region 260-270 rnp, where cr-tocopherylquinone has a high absorption maximum, indicates that at most only a small portion of the a-tocopherol was converted to the quinone. The products of the oxidation of ac-tocopherol in this system are probably mixtures of cu-tocopheroxide and reducing and nonreducing dimers which have been found to be present in the oxidation products of a-tocopherol in other systems (14). SUMMARY
The effects of temperature from -6’ to 30°C. and antioxidants upon the lipoxidase-catalyzed oxidation of aqueous sodium linoleate have been studied. An accurate analytical method for establishing the initial rates of the lipoxidase reaction has been described. The lipoxidase-catalyzed oxidation of linoleate was found to have a relatively low activation energy, 4300 cal./mole. Complete freezing of the lipoxidase system reduced the reaction rate to less than 1% of that found in the liquid system at the same temperature. All the phenolic antioxidants tested were found to inhibit the reaction. The greatest inhibitory effect was obtained with nordihydroguaiaretic acid, followed closely by propyl gallate; the effective concentrations were sufficiently low so that these two antioxidants might prove useful in protecting foods from this t,ype of oxidation. The biologically important antioxidant, cu-tocopherol, was rapidly oxidized in the lipoxidase system. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 11.
HOLMAN, R. T., ;irch. Biochem. 16, 403 (1947). GY~RC~Y, I?., AND TOMARELLI, R. RI., .T. Biol. Chem. 164, 317 (1944). STRAIN, H. H., J. Am. Chem. Sot. 63, 3542 (1941). KUNKEL, H. O., Arch. Biochem. 30, 306 (1951). HOLMAN, It. T., Trans. Third Conf. Riol. Antios., 1’. 131. Josiah Macy Jr. Foundation, New York 1948. THEORELL, H., BERGSTROM, S., ASI) .&c~:sox, A., Phnrm. .4&a Helv. 21, 318 (1946). BERGSTR~X, S., AKD HOLMAK, It. T., .~lricunces in E’ntpol. 8, 425 (1948). COSBY, E., AND SUMNER, J. B., rlrch. Biochem. 8, 259 (1945). SUMNER, J. B., AND SMITH, G. N., Arch. Biochem. 14, 87 (1947). SIZER, I. W., ANI! JOSEPHSOS, 13. S., Food Research 7, 201 (1942). TAPPEL, A. I,., BOYER, 1’. D., ASD I,I.NI)RER(:, W. 0.. .I. Biol. Chem. 199, 267 (1952). BOLLASD, J. I,., Proc. IZou. Sot. (London) A186, 218 (1946). KAUNITZ, H.. MD BEAVER. J. J., .J. Hiol. (“hem. 166, 6.53 (1911). Ii%OYER,
1’. D.,
.J. .tn~.
C’hcm.
,%c.
73.
734
(19511.