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Soya-bean lipoxygenase: An iron-containing dioxygenase
Biochimica et Biophysica Acta, 327 (1973) 32-35 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - P r i n t e d in The N e t h e r l a n d s
BBA
67O56
SOYA-BEAN L I P O X Y G E N A S E : AN I R O N - C O N T A I N I N G DIOXYGENASE*
H E N R Y W.-S. CHAN*"
University Chemical Laboratories, Lensfield Road, Cambridge, CB2 tE~V (Great Britain) (Received J u n e i5th, I973)
SUMMARY
Metal analysis by atomic absorption indicated the presence of one atom of iron in soya-bean lipoxygenase (linoleate:oxygen oxidoreductase, EC 1.13.11.12). Preincubation of the enzyme at pH 9 with chelating agents resulted in substantial inhibition. Tile results m a y indicate a role for the iron atom in lipoxygenase action.
INTRODUCTION
Lipoxygenase (linoleate:oxygen oxidoreductase, EC 1.13.11.12) was first isolated by Theorell et al. 1 who obtained a crystalline enzyme from tile soya-bean. More recent studies have shown the wider occurrence of this enzyme in the plant kingdom 2 a. While they differ in their specificity with respect to the position of oxygenation, all these enzymes catalyse tile formation of a hydroperoxide from linoleic acid. As they catalyse the incorporation of both atoms of a molecule of oxygen to the substrate, the enzymes may, therefore, be formally classified as dioxygenases. Unlike other enzymes in this class, however, no transition metal is believed to be associated with lipoxygenase. The requirement of a transition metal in dioxygenases may, at least, in part, originate from the high activation energy found in non-radical addition reactions between molecular oxygen and organic compounds. When conservation of spin angular m o m e n t u m is considered, the allowed product between molecular oxygen in its ground state (a triplet) and an organic molecule (other than a radical) in its ground state (a singlet) is a triplet--usually of high energy. It is possibly this "spin-barrier" which accounts for the high activation energy in reactions catalysed by dioxygenases. Tile formation of a complex between oxygen and a transition metal can be an effective means of lowering the activation energy and hence provide a pathway for catalysis. As part of a programme of investigation into the mechanism of dioxygenation, the question of the presence of a transition metal in lipoxygenase has been reexamined. * Results discussed herein were reported at the i I t h \Vorld Congress of the i n t e r n a t i o n a l Society for F a t Research held on 18-22 J u n e 1972, G6teborg, Sweden. ** P r e s e n t address: Agricultural Research Council, Food Research Institute, Colney Lane, Norwich, N O R 7OF, Great Britain.
SOYA-BEAN LIPOXYGENASE
33
MATERIALS AND METHODS
Enzyme purification Soya-bean lipoxygenase (Type I) was purchased from Sigma (London), Chemical Co. Ltd, S.W.6, Great Britain. The enzyme (2 g) was dissolved in 0.02 M phosphate buffer (20 ml, pH 6.2). After removal of insoluble material by centrifugation, it was applied onto a column (7 cm × 55 cm) of Sephadex G-2oo and eluted with the same buffer. Fractions containing the enzyme were pooled, concentrated by ultra-filtration and chromatographed on a column (2.5 cm × 7 cm) of DEAE-cellulose in 0.02 M phosphate buffer, pH 6.2, made up in de-ionised water. The enzyme was eluted as a single peak by a KC1 gradient (0-50 raM). Fractions containing the enzyme were pooled and concentrated by ultra-filtration. Disc-gel electrophoresis (7.5% polyacrylamide) showed a single protein band in gels (4 mm diameter) containing IO/~g and 50/zg of protein*.
Atomic absorption After the above purification, the lipoxygenase was prepared for metal analysis by atomic absorption as follows: a solution (I mg/ml) of the enzyme was dialysed against de-ionized water in an EDTA-treated dialysis bag for 60 h with five changes of water in a polyethylene container (500 ml). The dialysis residue was passed through a column (0.8 cm × 7 cm) of Sephadex G-25 in de-ionized water and lyophilised. The solid enzyme (IO rag) was dissolved in de-ionized water (5 rnl) to make up the solution for analysis. A fraction of the same volume as that containing the enzyme but from a region in the DEAE-cellulose chromatography that did not contain any ultraviolet absorbing material, was concentrated and treated as above and used as a control. Atomic absorption was measured on a Pye-Unicam SP 19oo double beam spectrophotometer. Standard solutions (0. 5 ppm and I.O ppm) of each metal to be determined were used to calibrate the instrument.
Enzyme assay and inhibition Lipoxygenase activity was measured by the increase in absorbance at 234 nm of a 0.2 mM solution of linoleic acid in 0.2 M Tris buffer (pH 9.0). I unit of enzyme causes an absorbance increase of o.ooi per rain at 25 °C with a final reaction volume of 3 ml and a light-path of i cm. The purified enzyme had an activity of 280 ooo units/rag. For the inhibition studies, the enzyme (5' lO-7 M) was incubated at 25 °C in o.I M Tris buffer (pH 9.0) containing the stated (Table II) molarity on inhibitor. Apart from KCN and EDTA which were added as aqueous solutions, the inhibitors were dispersed by addition as solutions in acetone, containing Tween 80 such that the final concentrations of acetone and Tween were 2% and o.ooi%, respectively. Two controls were used both containing the same concentration of the enzyme. In one, acetone and Tween were added to the above final concentrations. The activities of the enzyme were identical in the two controls.
The enzyme isolated has a pH maximum of 8.5 for linoleate oxygenation and corresponds to lipoxygenase i (Christopher et al.5).
34
RESULTS
H.W.-S.
AND
CHAN
DISCUSSION
I n the account of t h e i r original s t u d y , Theorell et al. 1 r e p o r t e d the presence of a small a m o u n t of iron ( ~ o . 3 mole per mole of enzyme) in lipoxygenase. Following the o b s e r v a t i o n t h a t chelating agents failed to i n h i b i t the enzyme, the iron was r e g a r d e d as an i m p u r i t y . Care has been t a k e n in t h e p r e s e n t i n v e s t i g a t i o n to ensure the p r o d u c t i o n of a purified s a m p l e of the e n z y m e as free from m e t a l c o n t a m i n a n t s as possible. A p a r t from being homogeneous in disc-gel eleetrophoresis, the e n z y m e used for analysis h a d a specific a c t i v i t y c o m p a r a b l e to t h a t o b t a i n e d p r e v i o u s l y b y isoelectric focussing 6. Steps were t a k e n to r e m o v e possible m e t a l c o n t a m i n a n t s prior to m e t a l analysis a n d a protein-free fraction from the final s t e p in the purification of the e n z y m e was t r e a t e d in parallel with the e n z y m e c o n t a i n i n g fraction a n d used as a control. Results o b t a i n e d (Table I) show t h a t iron is p r e s e n t in l i p o x y g e n a s e to the TABLEI A N A L Y S I S OF METALS IN S O Y A - B E A N L I P O X Y G E N A S E
Metal
Metal content in ppm Enzyme (2 mg/ml)
Control
A toms per mole of enzyme
Cu
o.o
o.o
o.o
Mn Fe Zn Mg
o.I i.o o.o o.o
o.o o.o o.o o.o
o.i 0.9 o.o o.o
e x t e n t of one a t o m per mole of enzyme. Moreover, of five m e t a l s analysed, only iron is p r e s e n t to an a p p r e c i a b l e degree. Confirmation of the presence of iron in lipoxygenase was o b t a i n e d b y t h e i n h i b i t i o n of the e n z y m e b y a v a r i e t y of chelating agents. Like Theorell et al. 1 we were u n a b l e to observe a n y inhibition of the e n z y m e b y these r e a g e n t s even a t high c o n c e n t r a t i o n s if t h e y are a d d e d at the t i m e of the enzyme assay. However, when the e n z y m e is p r e i n c u b a t e d at p H 9.o with the chelating agents, considerable i n h i b i t i o n is o b s e r v e d even a t low c o n c e n t r a t i o n s of the i n h i b i t o r s (Table II). Powerful chelating agents such as d i p h e n y l t h i o c a r b a z o n e , I , I o - p h e n a n t h r o l i n e a n d 2 , i ' - d i p y r i d y l are p a r t i c u l a r l y effective*. S o d i u m diethyld i t h i o c a r b o n a t e does n o t i n h i b i t as it is r a p i d l y d e c o m p o s e d a t this pH. The results discussed here s u p p o r t b u t do not p r o v e a role for t h e iron a t o m in catalysis in lipoxygenase. H o w e v e r , in view of t h e general i n v o l v e m e n t of t r a n s i t i o n m e t a l s in c a t a l y s i s in d i o x y g e n a s e s a n d the absence of o t h e r reasonable means of lowering the a c t i v a t i o n energies in d i o x y g e n a t i o n reactions, a role for the iron a t o m is s t r o n g l y i m p l i c a t e d . * These results appear to be at variance with those reported by Roza and Francke (see preceding article: Biochim. Biophys. Acta, 327, 24-3i ). This could be due to the use of much lower concentrations of the enzyme (5" Io-~ M) and dispersal agents for the solubilisation of the inhibitors in this study. The author thanks Ir Francke for communicating their results before publication.
SOYA-BEAN LIPOXYGENASE TABLE
35
II
]EFFECT OF INHIBITORS
ON L I F O X Y G ] E N A S ] E A C T I V I T Y A F T E R P R E I N C U B A T I O N
Inhibitor
Concentration (M)
Inhibition (%)
Diphenylthiocarbazone
Io -5
ioo 32 35 53 44
io -e
I, Io-Phenanthroline 2,2'-Dipyridyl 8-Hydroxyquinoline
IO-5 10-5 IO-~
KCN
10 -4
7I
EDTA
lO-4
60
ACKNOWLEDGEMENTS S u p p o r t f r o m a g r a n t f r o m t h e A g r i c u l t u r a l R e s e a r c h C o u n c i l is g r a t e f u l l y a c k n o w l e d g e d . T h e a u t h o r is also g r a t e f u l t o M r D. R. T h o m e r s o n f o r p e r f o r m i n g t h e atomic absorption analysis at Pye Unicam Ltd, Cambridge, Great Britain. REFERENCES I 2 3 4 5 6
Theorell, H., Holman, R. J. and ~_keson, A. (1947) Acta Chem. Scand. i, 571 Galliard, T. and Phillips, D. R. (I97 I) Biochem. J. 124, 431 Gardner, H. W. and Weisleder, D. (i97 o) Lipids 5, 678 Holden, M. (197 o) Phytochemistry 9, 5o7 Christopher, P. J., Pistorius, E. K. and Axelrod, B. (197 o) Biochim. Biophys. Acta 198, I2 Gatsinpoolas, N. (1969) Arch. Biochem. Biophys. I3I, 185
BBA
67O56
SOYA-BEAN L I P O X Y G E N A S E : AN I R O N - C O N T A I N I N G DIOXYGENASE*
H E N R Y W.-S. CHAN*"
University Chemical Laboratories, Lensfield Road, Cambridge, CB2 tE~V (Great Britain) (Received J u n e i5th, I973)
SUMMARY
Metal analysis by atomic absorption indicated the presence of one atom of iron in soya-bean lipoxygenase (linoleate:oxygen oxidoreductase, EC 1.13.11.12). Preincubation of the enzyme at pH 9 with chelating agents resulted in substantial inhibition. Tile results m a y indicate a role for the iron atom in lipoxygenase action.
INTRODUCTION
Lipoxygenase (linoleate:oxygen oxidoreductase, EC 1.13.11.12) was first isolated by Theorell et al. 1 who obtained a crystalline enzyme from tile soya-bean. More recent studies have shown the wider occurrence of this enzyme in the plant kingdom 2 a. While they differ in their specificity with respect to the position of oxygenation, all these enzymes catalyse tile formation of a hydroperoxide from linoleic acid. As they catalyse the incorporation of both atoms of a molecule of oxygen to the substrate, the enzymes may, therefore, be formally classified as dioxygenases. Unlike other enzymes in this class, however, no transition metal is believed to be associated with lipoxygenase. The requirement of a transition metal in dioxygenases may, at least, in part, originate from the high activation energy found in non-radical addition reactions between molecular oxygen and organic compounds. When conservation of spin angular m o m e n t u m is considered, the allowed product between molecular oxygen in its ground state (a triplet) and an organic molecule (other than a radical) in its ground state (a singlet) is a triplet--usually of high energy. It is possibly this "spin-barrier" which accounts for the high activation energy in reactions catalysed by dioxygenases. Tile formation of a complex between oxygen and a transition metal can be an effective means of lowering the activation energy and hence provide a pathway for catalysis. As part of a programme of investigation into the mechanism of dioxygenation, the question of the presence of a transition metal in lipoxygenase has been reexamined. * Results discussed herein were reported at the i I t h \Vorld Congress of the i n t e r n a t i o n a l Society for F a t Research held on 18-22 J u n e 1972, G6teborg, Sweden. ** P r e s e n t address: Agricultural Research Council, Food Research Institute, Colney Lane, Norwich, N O R 7OF, Great Britain.
SOYA-BEAN LIPOXYGENASE
33
MATERIALS AND METHODS
Enzyme purification Soya-bean lipoxygenase (Type I) was purchased from Sigma (London), Chemical Co. Ltd, S.W.6, Great Britain. The enzyme (2 g) was dissolved in 0.02 M phosphate buffer (20 ml, pH 6.2). After removal of insoluble material by centrifugation, it was applied onto a column (7 cm × 55 cm) of Sephadex G-2oo and eluted with the same buffer. Fractions containing the enzyme were pooled, concentrated by ultra-filtration and chromatographed on a column (2.5 cm × 7 cm) of DEAE-cellulose in 0.02 M phosphate buffer, pH 6.2, made up in de-ionised water. The enzyme was eluted as a single peak by a KC1 gradient (0-50 raM). Fractions containing the enzyme were pooled and concentrated by ultra-filtration. Disc-gel electrophoresis (7.5% polyacrylamide) showed a single protein band in gels (4 mm diameter) containing IO/~g and 50/zg of protein*.
Atomic absorption After the above purification, the lipoxygenase was prepared for metal analysis by atomic absorption as follows: a solution (I mg/ml) of the enzyme was dialysed against de-ionized water in an EDTA-treated dialysis bag for 60 h with five changes of water in a polyethylene container (500 ml). The dialysis residue was passed through a column (0.8 cm × 7 cm) of Sephadex G-25 in de-ionized water and lyophilised. The solid enzyme (IO rag) was dissolved in de-ionized water (5 rnl) to make up the solution for analysis. A fraction of the same volume as that containing the enzyme but from a region in the DEAE-cellulose chromatography that did not contain any ultraviolet absorbing material, was concentrated and treated as above and used as a control. Atomic absorption was measured on a Pye-Unicam SP 19oo double beam spectrophotometer. Standard solutions (0. 5 ppm and I.O ppm) of each metal to be determined were used to calibrate the instrument.
Enzyme assay and inhibition Lipoxygenase activity was measured by the increase in absorbance at 234 nm of a 0.2 mM solution of linoleic acid in 0.2 M Tris buffer (pH 9.0). I unit of enzyme causes an absorbance increase of o.ooi per rain at 25 °C with a final reaction volume of 3 ml and a light-path of i cm. The purified enzyme had an activity of 280 ooo units/rag. For the inhibition studies, the enzyme (5' lO-7 M) was incubated at 25 °C in o.I M Tris buffer (pH 9.0) containing the stated (Table II) molarity on inhibitor. Apart from KCN and EDTA which were added as aqueous solutions, the inhibitors were dispersed by addition as solutions in acetone, containing Tween 80 such that the final concentrations of acetone and Tween were 2% and o.ooi%, respectively. Two controls were used both containing the same concentration of the enzyme. In one, acetone and Tween were added to the above final concentrations. The activities of the enzyme were identical in the two controls.
The enzyme isolated has a pH maximum of 8.5 for linoleate oxygenation and corresponds to lipoxygenase i (Christopher et al.5).
34
RESULTS
H.W.-S.
AND
CHAN
DISCUSSION
I n the account of t h e i r original s t u d y , Theorell et al. 1 r e p o r t e d the presence of a small a m o u n t of iron ( ~ o . 3 mole per mole of enzyme) in lipoxygenase. Following the o b s e r v a t i o n t h a t chelating agents failed to i n h i b i t the enzyme, the iron was r e g a r d e d as an i m p u r i t y . Care has been t a k e n in t h e p r e s e n t i n v e s t i g a t i o n to ensure the p r o d u c t i o n of a purified s a m p l e of the e n z y m e as free from m e t a l c o n t a m i n a n t s as possible. A p a r t from being homogeneous in disc-gel eleetrophoresis, the e n z y m e used for analysis h a d a specific a c t i v i t y c o m p a r a b l e to t h a t o b t a i n e d p r e v i o u s l y b y isoelectric focussing 6. Steps were t a k e n to r e m o v e possible m e t a l c o n t a m i n a n t s prior to m e t a l analysis a n d a protein-free fraction from the final s t e p in the purification of the e n z y m e was t r e a t e d in parallel with the e n z y m e c o n t a i n i n g fraction a n d used as a control. Results o b t a i n e d (Table I) show t h a t iron is p r e s e n t in l i p o x y g e n a s e to the TABLEI A N A L Y S I S OF METALS IN S O Y A - B E A N L I P O X Y G E N A S E
Metal
Metal content in ppm Enzyme (2 mg/ml)
Control
A toms per mole of enzyme
Cu
o.o
o.o
o.o
Mn Fe Zn Mg
o.I i.o o.o o.o
o.o o.o o.o o.o
o.i 0.9 o.o o.o
e x t e n t of one a t o m per mole of enzyme. Moreover, of five m e t a l s analysed, only iron is p r e s e n t to an a p p r e c i a b l e degree. Confirmation of the presence of iron in lipoxygenase was o b t a i n e d b y t h e i n h i b i t i o n of the e n z y m e b y a v a r i e t y of chelating agents. Like Theorell et al. 1 we were u n a b l e to observe a n y inhibition of the e n z y m e b y these r e a g e n t s even a t high c o n c e n t r a t i o n s if t h e y are a d d e d at the t i m e of the enzyme assay. However, when the e n z y m e is p r e i n c u b a t e d at p H 9.o with the chelating agents, considerable i n h i b i t i o n is o b s e r v e d even a t low c o n c e n t r a t i o n s of the i n h i b i t o r s (Table II). Powerful chelating agents such as d i p h e n y l t h i o c a r b a z o n e , I , I o - p h e n a n t h r o l i n e a n d 2 , i ' - d i p y r i d y l are p a r t i c u l a r l y effective*. S o d i u m diethyld i t h i o c a r b o n a t e does n o t i n h i b i t as it is r a p i d l y d e c o m p o s e d a t this pH. The results discussed here s u p p o r t b u t do not p r o v e a role for t h e iron a t o m in catalysis in lipoxygenase. H o w e v e r , in view of t h e general i n v o l v e m e n t of t r a n s i t i o n m e t a l s in c a t a l y s i s in d i o x y g e n a s e s a n d the absence of o t h e r reasonable means of lowering the a c t i v a t i o n energies in d i o x y g e n a t i o n reactions, a role for the iron a t o m is s t r o n g l y i m p l i c a t e d . * These results appear to be at variance with those reported by Roza and Francke (see preceding article: Biochim. Biophys. Acta, 327, 24-3i ). This could be due to the use of much lower concentrations of the enzyme (5" Io-~ M) and dispersal agents for the solubilisation of the inhibitors in this study. The author thanks Ir Francke for communicating their results before publication.
SOYA-BEAN LIPOXYGENASE TABLE
35
II
]EFFECT OF INHIBITORS
ON L I F O X Y G ] E N A S ] E A C T I V I T Y A F T E R P R E I N C U B A T I O N
Inhibitor
Concentration (M)
Inhibition (%)
Diphenylthiocarbazone
Io -5
ioo 32 35 53 44
io -e
I, Io-Phenanthroline 2,2'-Dipyridyl 8-Hydroxyquinoline
IO-5 10-5 IO-~
KCN
10 -4
7I
EDTA
lO-4
60
ACKNOWLEDGEMENTS S u p p o r t f r o m a g r a n t f r o m t h e A g r i c u l t u r a l R e s e a r c h C o u n c i l is g r a t e f u l l y a c k n o w l e d g e d . T h e a u t h o r is also g r a t e f u l t o M r D. R. T h o m e r s o n f o r p e r f o r m i n g t h e atomic absorption analysis at Pye Unicam Ltd, Cambridge, Great Britain. REFERENCES I 2 3 4 5 6
Theorell, H., Holman, R. J. and ~_keson, A. (1947) Acta Chem. Scand. i, 571 Galliard, T. and Phillips, D. R. (I97 I) Biochem. J. 124, 431 Gardner, H. W. and Weisleder, D. (i97 o) Lipids 5, 678 Holden, M. (197 o) Phytochemistry 9, 5o7 Christopher, P. J., Pistorius, E. K. and Axelrod, B. (197 o) Biochim. Biophys. Acta 198, I2 Gatsinpoolas, N. (1969) Arch. Biochem. Biophys. I3I, 185