54
BIOCHIMICA ET BIOPHYSICAACTA
BBA 66691
ISOLATION OF A T H I R D ISOENZYME OF SOYBEAN LIPOXYGENASE"
J O H N P. C H R I S T O P H E R , E L F R I E D E K. PISTORIUS &ND B E R N A R D A X E L R O D
Department of Biochemistry, Purdue University, Lafayette, Ind. (U.S.A.)
(Received April ioth, 1972)
SUMMARY
I. A third isoenzyme of soybean lipoxygenase (linoleate: oxygen oxidoreductase, EC 1.13.I.13) has been purified to essential homogeneity by Sephadex DEAEA5o column chromatography and isoelectric focusing.
2. The enzyme differs from the two previously described pure isoenzymes
(lipoxygenases-i and -2) 1 by the following criteria: elution profile from DEAESephadex, isoelectric point, pH activity profile, and the effect of Ca 2+ on activity.
3. The new isoenzyme also shows an anomalous, inverse dependence of activity
on enzyme concentration.
INTRODUCTION
The possible occurrence of multiple forms of soybean lipoxygenase (linoleate:
oxygen oxidoreductase, EC I.I3.I.I3) has been intimated by several independenl
observations. Smith 2 and Sumner and Dounce 3, employing methyl linoleate and
cottonseed oil as substrates, respectively, reported a pH optimum of 6. 5 for the lipoxygenase activity of crude soybean extracts. Holman 4 showed that crystalline lipoxygenase had a pH optimum of 9.o with sodium linoleate as substrate.
In 1947, Kies 5, utilizing the coupled destruction of carotene as an assay, encountered anomalies which suggested the complex nature of soybean lipoxygenase
Employing a partially purified extract and trilinolein and linoleic acid as substrates
Koch et al. 6 obtained data which could best be explained by the existence of two enzymes. In 1967, Gusset al. 7 suggested that four lipoxygenases existed in aqueom
extracts of soybeans on the basis of disc gel electrophoresis followed by a specific
stain for enzyme activity.
More recently, three groups have reported on the isolation and partial characterization of an isoenzyme of soybean lipoxygenase. Investigating a guaiacol-linoleic acid hydroperoxide oxidoreductase activity in soybeans, Schormfiller el al. s
isolated two such activities. Both were homogeneous on cellulose acetate electrophoresis and also possessed lipoxygenase activity. The latter was not further charac* This is Journal Paper No. 4716 of the Purdue University Agricultural Experimem
Station.
Biochim. Biophys. Acta, 284 (1972) 54-62
THIRD ISOENZYME OF SOYBEAN LIPOXYGENASE
55
terized. Yamamoto et al. 9 have reported the isolation of an isoenzyme of lipoxygenase
which was distinct from the classical enzyme 1° in electrophoretic mobility, pH
profile, and response to calcium ions. Christopher et al. 1 have also reported the isolation of an ioenzyme (lipoxygenase-2) and established its identity as different from
the Theorell enzyme (hereafter referred to as lipoxygenase-I) on the basis of pH
profile, substrate specificity, electrophoretic mobility, and heat stability.
We now report on the isolation and partial characterization of a third isoenzyme
of lipoxygenase which has been shown to be different from lipoxygenases-I and -2.
MATERIALS AND METHODS
Materials
Soybeans of Hawkeye variety (197o crop) were used to prepare the enzymes.
Linoleic acid was obtained from Hormel Inst. (U. of Minn.), Sephadex DEAE-Aso
and G-I5O from Pharmacia, and isoelectric focusing equipment from LKB. Commercial lipoxygenase was purchased from Seravac.
Assays
Lipoxygenase activities were determined using a Clark oxygen electrode (Yellow Springs) in a Gilson Medical Electronics Oxygraph, Model KM. The reaction
vessel had a volume of 1. 7 ml. Values for oxygen concentrations n in solution were
not corrected for the effect of ionic solutes. One unit of enzyme activity corresponded
to the consumption of I/,mole 02 per rain. Assays were carried out under one of the
following conditions.
Method I. Samples were assayed at 15 °C and at two pH values (6.8 and 9.0)
using linoleic acid dispersed in Tween 20 (modification of Surrey's substrate) 12. At
pH 9.0, mixtures contained 165 mM sodium borate and 1.23 mM linoleic acid. At
pH 6.8, assays were performed with 165 mM sodium phosphate and 1.23 mM linoleic
acid, in the absence or presence of Ca 2+ (0.59 mM).
Method I I . Assays for pH optimum study were performed at 15 °C using linoleic acid dispersed in Tween 20. Reaction mixtures contained 1.23 mM linoleic acid
and 50 mM buffer of constant ionic strength (0.2).
Disc gel electrophoresis
Electrophoresis of protein was done on 7% acrylamide gels at pH 4.3, using
the method of Reisfeld et al. 13. A 25% glycerol solution was substituted for the stacking gel. Runs were performed at 5 °C for 2 h at a constant current of 3 mA per tube.
Protein was stained with Amido Schwarz and gels were scanned at 6oo nm.
Protein determination
Protein concentrations were calculated from absorbance values obtained at
280 nm. Measurement of the dry weight of purified lipoxygenase gave a value of 0.7
mg protein per ml per absorbance unit.
Elution profile on Seph~dex G-z5o
An indication of molecular weight was obtained by gel filtration at room temperature. A column (2.5 cm × 48 cm) of Sephadex G-I5O was poured and equiliBiochim. Biophys. Acta, 284 (I972) 54-6~
j.P. CHRISTOPHERel al,
56
brated with 0.05 M sodium phosphate (pH 6.8) containing o.I5 M NaC1. Void volume
was determined with 2 ml of 0.2% Dextran Blue. Protein samples (3-4 rag) were
applied and eluted with the above buffer. The flow rate was adjusted to 3 ° ml/h
and fractions of 2.5 ml were collected. Fractions were assayed for activity at pH
values 6.8 and 9.0.
EXPERIMENTAL
Purification
Hexane-defatted soybean meal (75 g) was extracted with 750 ml of water at
room temperature for i h with stirring. The suspension was passed through one laye~
of cheesecloth and the resulting filtrate centrifuged at 16 ooo × g for IO min. The
precipitate was discarded and the supernatant treated as described below. All subsequent steps were performed at 0-5 °C and centrifugations at I6 ooo × g for io rain.
Sodium phosphate buffers were employed unless otherwise specified. To the supernatant (600 ml) from above, 5° m l of a 0.58 M CaC12 solution were added. The resulting precipitate was centrifuged and discarded. The supernatant (60o ml), which
~.
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~ ACTIVITY,pH9.0
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30
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,~
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20
q//'
40
60
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80
I
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IO0 l120 140 160 180
200
FRACTION NUMBER
Fig. I. S e p h a d e x DILAE A5o column c h r o m a t o g r a p h y . A coluinn (4 cm x 38 cm) was packe(!
with D E A E - A 5 o and equilibrated with o.o2 M sodium p h o s p h a t e (pH 6.8). The e n z y m e sample
was applied in the same buffer and elution was effected (7 ° ml/h) w i t h a linear gradient formed
f r o m equal volumes of o.o2 M and o.22 M sodium p h o s p h a t e (pH 6.8). Fractions of 17 ml wer~
collected and protein c o n t e n t determined by a b s o r b a n c e at 28o nm. Activity was assayed by Met h o d I in the presence of o.59 inM Ca 2+.
Biochim. Biophys. Acta, 284 (1972) 54-62
THIRD ISOENZYME OF SOYBEAN LIPOXYGENASE
57
constituted the crude extract, was made to 75% saturation with respect to (NH4)2SO4
by the addition of 31o g of the solid salt. The p H was adjusted to 6.8 ± 0.2 with 2 M
NaOH. The precipitated protein was centrifuged and then dissolved in o.o 5 M buffer
(pH 6.8) to give a volume of 145 ml. This protein solution was dialyzed overnight
against 4 ° volumes of o.oi M buffer (pH 6.8) containing 0.5 mM Ca a+. The dialysate
from above was collected and centrifuged. The inactive precipitate was discarded,
and the supernatant (145 ml) was successively fractionated with solid (NH4)2SO 4 to
fractions corresponding to 0-25%, 25 55%, and 55-75~o saturation. The bulk of the
desired activity was in the 25-55% fraction which was dissolved in 7 ° ml 0.05 M
buffer (pH 6.8) and dialyzed overnight against 4 ° volumes of 0.02 M buffer (pH 6.8)
containing 0.5 mM Ca 2+.
After eentrifugation, the 25-55~g fraction (73 ml and 730 A2s0 n m units) was
subjected to chromatography on a column of DEAE-Sephadex. The elution pattern
obtained is illustrated in Fig. I. The first enzyme peak to emerge represents the new
isoenzyme (lipoxygenase-3), the second, lipoxygenase-2 and the last, lipoxygenase-i.
Fractions 45-60 were pooled.
A portion (50 ml) of these pooled fractions was dialyzed overnight against
20 volumes of deionized water to reduce salt content. This solution was subjected
to isoelectric focusing in an L K B IiO ml column at 4 °C, according to the method oi
Vesterberg and Svensson 14. The sucrose gradient was prepared with the aid of a
gradient mixer. The protein solution (15.2 A~80 nm units) was incorporated into the
light solution. L K B ampholytes (40%) of p H range 3-1o were employed at a final
column concentration of 1% . The bottom electrode served as the anode. The column
&
A
pH
~.
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~.
0
PROTEIN
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13
12
I.I
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FRACTION
20
24
28
32
36
40
NUMBER
Fig. 2. I s o e l e c t r i c focusing. I s o e l e c t r i c f o c u s i n g w a s p e r f o r m e d on a n L K B 82Ol a p p a r a t u s a t
4 °C for 41 h. The p r o t e i n s a m p l e w a s focused in a p H g r a d i e n t of 3-1o a t a final a m p h o l y t e concent r a t i o n of I ~o. F r a c t i o n s of 3 m l were collected. P r o t e i n w a s d e t e r m i n e d b y a b s o r b a n c e a t 28o n m
a n d a c t i v i t y b y M e t h o d I in t h e p r e s e n c e of 0.59 mM Ca 2+. T h e p H of e a c h f r a c t i o n w a s m e a s u r e d
a t 23 °C.
Biochim. Biophys. Mcta, 284 (1972) 54-62
j.P. CHRISTOPHERet al.
58
was charged at a flow rate of 3 ml/min and emptied at 1.5 ml/min. Voltage was maintained at 200 V for 17 h and then increased to 30o V for 24 h; initial current was
6.8 mA, Fractions of 3 ml were collected. The elution profile obtained on isoelectric
focusing of the material represented by the first peak of the D E A E column exhibited
one peak of lipoxygenase activity (see Fig. 2), with an isoelectric point of 6.15.
Fractions 16-18 were combined. Ampholytes were removed by adding 0.3 g NaC1 to
the sample (9 ml) and dialyzing this solution overnight against 75 volumes 0.05 M
buffer, pH 6.8, containing o.15 M NaC1. The dialysate was collected, 0.3 g NaC1 was
added, and the solution dialyzed overnight against the above buffer. The dialysate
was removed and dialyzed against 75 volumes of fresh 0.05 M buffer (pH 6.8) for
48 11. Protein was concentrated by ultrafiltration employing a Io-ml capacity Amicon
cell equipped with a PM-Io filter. Ultrafiltration was performed at room temperature
under 4 ° lb/ineh 2 nitrogen. This concentrated protein solution was employed for subsequent studies. The purification of lipoxygenase- 3 is summarized in Table I. An
explanation for the discrepancy in the values for total activity at pH 6.8 is presented
below (see activity as a function of enzyme concentration).
TABLE I
SUMMARY" OF PURIFICATION
Step
Crude extract
(NH4)~SO4 concentrate
(NH4)2SO, fractionation
Sephadex DEAE-A5o
Isoelectric focusing
Vol, (ml)
600
145
73
255
9
Total protein
(A~8o nm units)
ii 400
I 600
73°
76.5
4-4
Total activity (units)
pH 6.8"
pH 9.o
12 200
18 700
14 ioo
84I (663)*"
5 t (I8O)**
13 ooo
8 ooo
6 500
--
* Values refer to lipoxygenase-3 units, assuming linearity between enzyme content and O~
consumption.
** Values in parentheses refer to lipoxygenase-3 units as defined in the text.
RESULTS
Disc gel electrophoresis
Electrophoresis at pH 4.3 revealed the presence of one major band (RF o.3o7)
and two trace bands (RE values 0.240 and o.173 ). Comparison of appropriate peak
areas (Fig. 3) indicated that the two trace bands together comprised less than 5%
of the total protein.
Additional experiments, subsequent to the completion of this work have shown
that chromatography on a column of Sephadex CM separates the material corresponding to the band of RE 0.307 from the trace impurities. Furthermore, the former
was shown to possess lipoxygenase activity whereas the latter are inactive.
p H profile
The enzyme activity as a function of pH, using linoleic acid as substrate (see
Assay: Method liT) is shown in Fig. 4. Activity was seen at all pH values tested except
pH 9.0. The p H optimum is very broad under these conditions.
Biochim. Biophys. Acta, 284 (1972) 54-62
THIRD ISOENZYME OF SOYBEAN LIPOXYGENASE
59
Q
I
__m
®
ABSORBANCE
AT 6 0 0 nm
Fig. 3. Polyacrylamide gel electrophoresis. Lipoxygenase-3 (22/~g) was layered on the gel surface
in a 25% glycerol solution. Electrophoresis (pH 4-3) was performed in 7% gel for 2 h at 5 °C
and at a constant current of 3 mA per tube. Protein was stained with a solution of Amido-Schwarz,
and gel was scanned at 6o0 nm after destaining electrophorctically.
o
~ ACETATE
~ PHOSPHATE
-~ BORATE
•"-
I0
r~
9
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8
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4.0
4.5
5D
5.5
6.0
6.5
7.0
7.5
80
8.5
9.0
pH
Fig. 4. Activity of lipoxygenase-3 toward linoleic acid as a function of pH at constant ionic strength
Assays were performed by Method II and 18/~g of enzyme were employed per test. Average values
of activity were plotted in regions of overlapping buffers.
Elution profile on Sephadex G-zSO
Simultaneous gel filtration of lipoxygenase-3 and a commercial lipoxygenase,
as a source of lipoxygenase-I, resulted in the elution curve shown in Fig. 5. The activities at pH 9.0, characteristic of lipoxygenase-I, and pH 6.8, characteristic of lipoxygenase-3, were both eluted at the same Ve/Vo ratio. The same result was also
Biochim. Biophys. Acta, 284 (1972) 54-62
6o
j.P. CHRISTOPHER et al.
=
~. DEXTRAN BLUE
0 ACTIVITY, pH 6.8
~ ACTIVITY, pH 9.0
0
~,
1.6
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ELUTION
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Fig. 5. E l u t i o n profile on S e p h a d e x G - i s o of lipoxygenase-3. A S e p h a d e x G-I5O c o l u m n (2. 5 c m ×
48 cm) w a s e q u i l i b r a t e d w i t h o.o 5 M s o d i u m p h o s p h a t e (pH 6.8) c o n t a i n i n g o. 15 M NaC1. L i p o x y g e n a s e s - I a n d -3 (3-4 m g each) were applied either in a d m i x t u r e or s e p a r a t e l y a n d eluted with
t h e a b o v e buffer. F r a c t i o n s (3 ml) were a s s a y e d for a c t i v i t y b y M e t h o d I in t h e a b s e n c e of Ca 2÷.
obtained when the enzymes were chromatographed separately. Thus, it was concluded
that lipoxygenase-I and lipoxygenase-3 had similar Stokes radii. Lipoxygenase-I has
been reported to have a molecular weight of about IOO ooo based on sedimentation
velocity ~°, equilibrium sedimentation 1'~, and gel filtration 1G.
Effect of Ca 2+ on activity
The response of activity to increasing concentrations of Ca 2+ is illustrated in
Fig. 6. Ca 2+ did not stimulate activity. Indeed, Ca 2+ was inhibitory at all concentrations tested.
Activity as a function of enzyme concentration
The striking deviation from a linear relationship between the quantity of lipoxygenase-3 taken for assay and the rate of O~ consumption is illustrated in Fig. 7Specific activity varied more than 3.5-fold over a 2o-fold range in enzyme concentration. The experimental points were obtained using a series of enzyme concentrations under standard assay conditions during which the rate of O~ utilization was proportional to reaction time. A unit of lipoxygenase-3 was defined arbitrarily as ten
times the quantity of enzyme catalyzing the consumption of precisely o.Io #mole of
O~ per min under the standard assay conditions.
Biochim. Biophys. Acta, 284. (i972) 54-6:z
61
THIRD ISOENZYME OF SOYBEAN LIPOXYCENASE
i
i
I
0.1
I
0.2
i
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0.30
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025
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06
CALCIUM
I
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ION CONCENTRATION ( m M )
I
0
I
I
I
J
I
[
I
I
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LIPOXYGENASE - 3 UNITS
I
2
ENZYME
I
3
PER
I
4
I
,5
[
0.9
I
6
10
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ASSAY(Aze 0 UNITS x IO2)
Fig. 6. Effect of calcium ion oi1 activity. Assays were performed b y Method I at p H 6.8 u n d e r
v a r y i n g c o n c e n t r a t i o n s of Ca 2+ and 18 ,ug of lipoxygenase-3 per assay.
Fig. 7. Activity as a function of enzyme concentration. V a r y i n g c o n c e n t r a t i o n s of lipoxygenase-3
were enlployed. Assays were p e r f o r m e d according to Method I at p H 6.8 in the absence of Ca 2+.
DISCUSSION
The newly-isolated isoenzyme, lipoxygenase-3, appeared to have a molecular
weight of ioo ooo as judged by gel filtration, the same value as found for the other
two isoenzymes. However, lipoxygenase-3 exhibited a number of differences in other
respects.
(I) Chromatographic behavior on DEAE Sephadex clearly differentiated between all three isoenzymes.
(2) Different isoelectric points were found for each isoenzyme : 5.68 for ]ipoxygenase-i, 6.25 for lipoxygenase-2 (unpublished results) and 6.15 for lipoxygenase-3.
The value for lipoxygenase-I agreed well with the pI of 5.65 obtained by Catsimpoolas 17.
(3) The pH-activity profiles of each were significantly different under similar
assay conditions. Lipoxygeanses-I and -2 exhibited pH optima of 9.5 and 6.5, respectively 1. Lipoxygenase-3, however, had a broad pH-activity profile throughout
the pH range 4.5-9.0 and showed no activity at pH 9.
(4) The specific activities of lipoxygenases-I and -2 were not dependent on enzyme concentration in contrast to lipoxygenase- 3.
(5) Lipoxygenase-3 could be distinguished from lipoyxgenase-2 with respect to
the effect of Ca 2+ on activity. Ca 2+ stimulated the activity of lipoxygenase-29 but
inhibited the activity of lipoxygenase-3 under similar conditions.
It is clear that at least three isoenzymes, and possibly four:, of lipoxygenase
occur in soybean extracts. The molecular basis for these differences is unknown. The
Biochim. Biophys. Acta, 284 (1972) 54-6~
62
J.P.
CHRISTOPHER el a l
possibility that these multiple forms might be hybridized dimers of monomerie spe
cies m a y deserve consideration since Stevens et al. 15 have indicated that lipoxygenase.
I consists of two subunits of equal molecular weight. However, we (unpublished re
sults) have observed no dissociation of lipoxygenase-I on electrophoresis in sodiundodecylsulfate gels after treatment of protein with mercaptoethanol.
The reason for tile striking non-linearity of specific activity as a function o:
enzyme concentration is not known. It cannot be the result of enzyme inactivatior
nor depletion of substrate because the rates of oxygen consumption are lineal
throughout the reaction period on which tile assay is based. These aspects of tile problem as well as other differences between the isoenzymes are currently under investi
gation.
REFERENCES
i
2
3
4
5
6
7
8
9
io
ii
i2
13
14
15
16
17
J. Christopher, E. P i s t o r i u s a n d B. Axelrod, Biochim. Biophys. Acta, 198 (197 o) 12.
G. N. S m i t h , Arch. Bioehem. Biophys., 19 (1948) 133.
J- ]3. S t u n n e r a n d A. L. D o u n c e , Enzymologia, 7 (1939) 13o.
R. T. H o l m a n , Arch. Bioehem. Biophys., 15 (1947) 403 •
M. W. Kies, Fed. Proc., 6 (1947) 267R. B. Koch, B. S t e r n a n d C. G. Ferrari, Arch. Biochem. Biophys., 78 (1958) 165.
P. L. Guss, T. R i c h a r d s o n a n d M. A. S t a h m a n n , Cereal Chem., 44 (1967) 607.
J. Schornlt~ller, J. W e b e r , B. H6xer, a n d "vV. Grosch, Z. Lebensm.-Unters.-Forsch. 139 (1969'
357.
A. Y a m a m o t o , K. Y a s u m o t o a n d H. M i t s u d a , dgric. Biol. Chem., 34 (197 o) 1169.
H. Theorell, R. T. H o h n a n a n d 2~. 2~keson, Acta Chem. Scan&, I (1947) 571.
C. D. H o d g m a n , Handbook of Chemistry and Physics, Chemical R u b b e r , Clevelaml, Ohio
36th edn, 1954 1955, p. 161o.
K. Surrey, Plant Physiol., 39 (1964) 65.
R. A. Reisfeld, U. J. Lewis a n d D. E. Williams, Nature, 195 (I962) 281.
O. V e s t e r b e r g a n d H. Svensson, dcta Chem. Scan&, 20 (1966) 820.
F. C. Stevens, D. M. B r o w n a n d E. L. S m i t h , Arch. Biochem. Biophys., 136 (197 o) 413 .
D. H. S c h r o e d e r Some Properties of Soybean Lipoxidase, P h . D . Thesis, P u r d u e University
West L a f a y e t t e , Ind., 1968.
N. C a t s i m p o o l a s , Arch. Biochem. Biophys., 131 (1969) 185.
Biochim. Biophys. Acta, 284 (1972) 54-62