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Some properties of soybean lipoxygenase
ARCHIVES
OF
BIOCHEMISTRY
AND
Some FRITS Department
136, 413-421 (1970)
BIOPHYSICS
Properties
of Soybean
C. STEVENS?, DOUGLAS
o-f Biological
Chemistry, Received
UCLA
August
bipoxygenase’
M. BROWN,
School of Medicine, California 900.??4
11, 1969; accepted
AND EMIL University
November
L. SMITH3
of California,
Los ilngeles,
18, 1969
Commercial soybean lipoxygenase (EC 1.99.2.1) was purified by ammonium sulfate fractionation, gel filtration on Sephadex G-150, and chromatography on DEAEcellulose. The purified protein was essentially homogeneous as judged by acrylamide gel electrophoresis and ultracentrifugation. The molecular weight is 108,000 as determined by the sedimentation-equilibrium method. The amino acid composition was determined, and it was shown that the protein contains four residues of free sulfhydryl groups and four residues of half-cystine per molecule. Treatment of the protein with guanidine hydrochloride or sodium dodecyl sulfate produces dissociation. Present evidence indicates that the protein is composed of two subunits of 54,090 molecular weight.
Lipoxygenase (EC 1.99.2.1, linoleate:oxygen oxidoreductase) catalyzes the oxidation by moleculas oxygen of cis-methylene interrupted unsaturated fatty acids and their esters to respective hydroperoxides (1) : R-cH=CH--CH,-CH=CHR,
+ 0,
R-CH=CH-CH=CH-CH-R,
A
H09
The enzyme was discovered through its concurrent oxidation0 of carotene (2). Theorell, Holman, and Akeson (3) obtained a crystalline preparation from soybeans which was homogeneous, as judged by ultracentrifugation and moving boundary electrophoresis, and had an estimated molecular weight of 102,000 and an isoelectric point at pH 5.4. Holman (4) found that lipoxygenase 1 This investigation was aided by Grant GM 11061 from the National Institute of General Medical Sciences, United States Public Health Service. 2 Present address: Department of Biochemistry, Faculty of Medicine, University of Manitoba, Winnipeg 3, Manitoba, Canada. 3 To whom inquiries and requests for reprints should be addressed.
is not inhibited by pyrophosphate, fluoride, cyanide, azide, p-chloromercuribenzoate, mercury ions, or diethyldithiocarbamic acid, and concluded that the activity of the enzyme is not dependent on metal ions or sulfhydryl groups. Using the then available techniques of paper chromatography and microbiological assays, Holman et ~2. (5) also determined the amino acid composition of lipoxygenase. Tappel, Boyer, and Lundberg (6) studied the reaction mechanism of the enzyme and on the basis of their results proposed a simple mechanism involving formation of a biradical from linoleate and oxygen on the surface of the enzyme. The biradical may accept electrons from antioxidants or may react to give the conjugated linoleate peroxide. The extreme specificity of lipoxygenase was recently demonstrated by Hamberg and Samuelson (7). Walker (8) demonstrated by EPR spectroscopy the existence of free radicals during the course of linoleate peroxidation by the enzyme. Dolev and co-workers (9-11) showed in labeling experiments that the oxygen incorporated into the hydroperoxide product is derived from molecular oxygen and not from water.
414
STEVENS,
BROWN,
It should be noted that many of the studies on lipoxygenase have been carried out with commercial preparations, which were found to be heterogeneous in the present investigation. While our work was in progress Rfitsuda et al. (12) reported studies on the purified enzyme and investigated the effect of saturated monohydroalcohols on the reaction in order to elucidate the role of hydrophobic regions of enzyme on the binding of the substrate (13). Allen (14) has also reported an improved method for preparation of the enzyme. No attempts have been made recently to characterize the enzyme as a protein by modern methods, and the nature of the catalytic site is still not understood. We undertook the present study in order to obtain information concerning the subunit of this unstructure and the composition usual enzyme. EXPERIMENTAL
PROCEDURE
The starting material was a commercial preparation (Lipoxidase Lx 6EA) of soybean lipoxygenase obtained from the Worthington Biochemical Corporation, Freehold, New Jersey. A preparation (No. 389093) purchased from Fluka, A. G., Buchs, S. C., Switzerland, was also investigated. Chemicals. Sephadex G-150 (40-120 p) was obtained from Pharmacia Fine Chemicals, Inc., Nutley, New Jersey and DEAE-cellulose with an exchange capacity of 0.75 meq/g (Cellex-D No. 4551) was purchased from Bio-Rad Laboratories, Richmond, California. Reagent grade iodoacetic acid was recrystallized from petroleum ether (60110”). Guanidine hydrochloride was reagent grade (J. T. Baker Chemical Co.) and was washed with acetone to remove yellow color. The Ellman reagent, 5,5’-dithiobis-(2.nitro-benzoic acid) was obtained from the Aldrich Chemical Company, Inc., Milwaukee, Wisconsin, and sodium dodecylsulfate was a commercial preparation (Sipon SD) of the American Acoolac Corporation, Baltimore, Maryland. All other chemicals used were reagent grade. Enzyme assays. Lipoxygenase activity was determined by the following method. One hundred microliters of chromatographically pure linoleic acid (Calbiochem) were dissolved in 15 ml of absolute ethyl alcohol, and water was added to make 25 ml of stock solution; this solution was made fresh every day. Immediately before use, 5 mi of substrate stock solution were diluted with 25 ml of 0.2 M sodium borate buffer at pH 9.0, and
AND
SMITH
the solution was oxygenated by bubbling gaseous oxygen through for a few minutes. The enzyme solution to be assayed was also diluted with 0.2 M sodium borate buffer at pH 9.0 to give an absorbance reading of 0.050 at 280 rnh. Two milliliters of the diluted substrate solution were pipetted into a I-cm silica glass cuvette, and at zero time 0.05-l ml of enzyme solution and buffer were added to make a total volume of 3 ml. The change in absorbance at 234 mp, produced by formation of hydroperoxide from the substrate, was measured as a function of time in a Zeiss M4QIII spectrophotometer. Readings were taken every 30 set for several minutes. One enzyme unit is defined as a change in absorbance (A) of 0.01 in 1 min. Specific activity of enzyme preparations are expressed as number of enzyme units per 0.01 absorbance unit at 280 rnM of the enzyme solution (Am). With very dilute enzyme solutions there was an initial lag period and the change in absorption at 234 mp became linear only after 1 min. All measurements were taken in the linear portion of the change in absorbance with time. Under these conditions enzyme activity was strictly proportional to protein concentration. Amino acid analyses. Samples of the purified protein were thoroughly dialyzed, first against 0.1 M KC1 and then against deionized water. After lyophilization, aliquots were hydrolyzed in threetimes glass-distilled 6 N HCl at 110” in sealed, evacuated tubes. Analyses were performed in duplicate after 24 and 70 hr of hydrolysis on the Spinco automatic amino acid analyzer by the method of Spackman, Stein, and Moore (15). Tryptophan was determined spectrophotometrically (16) and also by the calorimetric method of Spies and Chambers (17). The half-cystine content was determined after performic acid oxidation by the method of Moore (18). Total hexose was determined by the method of Tsugita and Akabori (19), as modified by Carsten and Pierce (20). Free sulfhydryl groups were determined by the method of Ellman (21)) as described by FernandezDiez, Osuga, and Feeney (22). Alternatively, sulfhydryl groups were estimated as S-carboxymethylcysteine by amino acid analysis after alkylation by the method of Crestfield, Moore, and Stein (23). Ultracentrifuge studies. Equilibrium experiments were performed with a Spinco Model E ultracentrifuge equipped with interference optics and constant temperature control (RTIC). Sedimentation velocity studies were run at 59,780 rpm Acrylamide gel electrophoresis. Acrylamide gel electrophoresis was performed in 7.5% gels in Tris-glycine buffer at pH 8.8, as described by
SOYBEAN
LIPOXYGENASE
Ornstein and Davis (24). Samples were applied to the top of the gel in buffer containing 15y0 sucrose.
415 TABLE
PURIFICATION
OF
SOYBEAN
I LIPOXYGENASE
RESULTS
Purijication. Figure 1 shows the acrylamide electrophoresis patterns of preparations of lipoxygenase obtained from Worthington and Fluka, respectively. Both preparations were heterogeneous by this technique. Sedimentation velocity centrifugation of Worthington lipoxygenase at pH 7.4 (0.035 M Verona1 + 0.065 M NaCl) revealed at least three overlapping peaks. Both commercial preparations had an identical specific activity of 49 enzyme units per absorbance unit. The Worthington preparation was purified as described below; results are summarized in Table I. Figure 2 shows the electrophoretic pattern in acrylamide gel at various stages of purification. A six-fold purification was achieved with a 50 to 60 % recovery of the original activity.
FIG. 1. Acrylamide gel electrophoresis of commercial lipoxygenase: left, 250 pg of Worthington lipoxygenase; right, 250 rg of Fluka lipoxygenase. The samples were run on 7.57” gels at a constant current of 5 mA per tube for 50 min in the cold. They were stained with amido schwarz.
Crude lipoxygenase (Worthington) (NH&SOa fractionation active fraction on 40-60% cut Gel filtration on Sephadex G-150 60% saturated (NH&S04 precipitate of active peak Chromatography on DEAE-cellulose 60% saturated (NH&SO4 precipitate of active peak
49
100
65
95
130
82
300
60
a The activity was measured as described in the Experimental section.
a. Ammonium sulfate fractionation. Lipoxygenase (8 g) was dissolved in 400 ml of 30% saturated ammonium sulfate at 4O and then brought to 40% saturation by addition of solid ammonium sulfate. After standing overnight at 4” the inactive precipitate was removed by centrifugation and discarded. The supernatant solution was brought to 60 % saturation by addition of solid ammonium sulfate and after standing overnight in the cold, the precipitate was collected by centrifugation. This fraction, accounting for almost all of the original activity and for approximately 80% of the original protein, was redissolved in 50 ml of 0.1 31 ammonium bicarbonate and dialyzed against three changes of 4 liters of the same buffer over a period of 48 hr. This solution was stable and showed no loss of activity over a period of more than a month in the cold. The treatment increased the specific activity of the enzyme from 49-65 enzyme units/absorbance unit. b. GelJiltration on Xephadex G-150. Approxmately 20-ml batches of the lipoxygenase solution in 0.1 M (NH,)HCO;( were applied at 4O to a 3.8 X 127-cm column of Sephadex G-150 (40-120 cl) equilibrated with the same buffer. Elution was performed with the same buffer under a hydrostatic pressure of approximately lo-20 cm of water. This gave a flow rate of 40-50 ml/hr, and 10 tubes per hour were collected with the
416
STEVENS,
BROWN, AND SMITH
TUBE
NUMBER
FIG. 3. Purification of ammonium sulfate-fractionated Worthington lipoxygenase on Sephadex G-150. The details are described in the text. The absorbance at 280 mc: (0) and the enzymatic activity (x) were determined on effluent fractions. The solid bar indicates the active fractions pooled.
FIG. 2. Acrylamide gel electrophoresis of lipoxygenase at various stages of purification. From left to right: 250 pg of Worthington lipoxygenase, 100 rg of the act,ive fraction after gel filtration on Sephadex G-150, 50 fig of the active fraction after chromatography on DEAE-cellulose, 100 fig of the active fraction after chromat,ography on DEAE-cellulose. The samples were run on 7.5y0 gels at a constant current of 4 mA per tube in the cold and stained with amido schwarz.
aid of an automatic fraction collector. l’rotein absorption at 280 rnp and enzymic activity were determined on alternate fractions. Figure 3 shows a typical elution pattern. Active fractions from consecutive runs on the same column were pooled and the protein was precipitated by bringing the solution to 60% saturation by addition of solid ammonium sulfate at 4’. This material had a specific activity of 130 enzyme units/ absorbance unit. Acrylamide gel electrophoresis (Fig. 2) showed that there were
two major and some minor components still present. c. Column chromatography on DEAE-cellulose. Further purification was achieved by chromatography on DEAE-cellulose at 4’ with a linear gradient from 0.02 M sodium phosphate at pH 7.5 to 0.02 M sodium phosphate at pH 7.5 + 0.5 M NaCl. The details of the run and a typical elution pattern are shown in Fig. 4. Since the enzyme was rather unstable under these conditions, the active fractions were pooled, as soon as they were located, and solid ammonium sulfate was added to 60% saturation. The purified enzyme could be kept at 4’ indefinitely under these conditions without noticeable loss of activity; however, it loses activity upon dialysis and lyophilization. The purified lipoxygenase had a specific activity of 300 units per absorbance unit and gave a single band on acrylamide gel electrophoresis at pH 8.8 (Fig. 2). Molecular weight determination. The molecular weight of purified lipoxygenase was determined by the high-speed sedimentation-equilibrium method of Yphantis (25). The partial specific volume (V) of the protein was calculated from its amino acid composition (see below) as 0.734. A sample of lipoxygenase was dialyzed overnight against 0.035 M sodium Verona1 buffer f 0.065 M NaCl at pH 7.4. A three-
SOYBEAN
TUBE
LIPOXYGENASE
NUMBER
4. Purification of lipoxygenase on DEAEcellulose. Approximately 160 mg of the active Sephadex G-150 fraction were dissolved in and dialyzed overnight against 0.02 M sodium phosphate buffer at pH 7.5. The sample (3 ml) was applied to a column of DEAE-cellulose (1.7 X 15 cm) equilibrated with the same buffer. A flow rate of 36 ml/hr was maintained with a Beckman Accuflow pump and 4-ml fractions were collected. Separation was achieved by passing through 100 ml of 0.02 M sodium phosphate buffer at pH 7.5, followed by a linear gradient which was established with 100 ml 0.02 M sodium phosphate buffer, pH 7.5 + 0.5 M NaCl.
FIG. 5. Sedimentation-equilibrium ultracentrifugation of soybean lipoxygenase. Plot of logarithm of the concentration (C) in fringes versus radial distance squared is shown at 22 hr after reaching a speed of 14,000 rpm. Concentration shown is 0.6 mg/ml .
channel equilibrium cell was filled with 0.4, 0.6, and 1.0 mg/ml protein solution and run at 14,000 rpm after overspeeding for 1 hr at 20,000 rpm. The temperature was maintained at 20’ throughout the run. The plates were read at each half fringe on the Gaertner microcomparator at 22 hr after attaining equilibrium speed. The calculations were programmed into a Monroe Epic 3000 computer. The results for the middle channel are shown in Fig. 5. The sample was found to contain two molecular components with the molecular weight at the meniscus M, = 108,000 f 2,000. The small amount (approximately 5 % from sedimentation-velocity determination reported below) of heavier material had a molecular weight greater than 188,000 and could represent some dimerization. Subunit structure of soybean lipoxygenase. For a sedimentation-equilibrium experiment a sample was dialyzed for 48 hr against 6 M guanidine hydrochloride containing 0.5 % mercaptoethanol at pH 7.7. Concentrations of 0.4, 0.6, and 0.8 mg/ml were placed into the three-ch.annel Yphantis-type cell, and
the run was performed for 20 hr at 24,630 rpm. The material was found to be heterogeneous, giving at the meniscus M, = 58,000 and at the bottom of the cell Mb = 112,000. There was also a small amount of unresolved heavier material at the bottom of the cell. It is evident that only partial dissociation of the protein wa.s obtained in this experiment. By the technique of sedimentation velocity, three samples were studied at a concentration of 10 mg/ml: (1) in 0.035 31 Verona1 + 0.64 M NaCl at pH 7.4; (2) in the same buffer + 0.5% sodium dodecyl sulfate; (3) in the same buffer + 1% sodium dodecyl sulfate. These samples were run at 59,780 rpm at 20”. Pictures were taken in all three cases starting at 28 min after attaining speed and then at 8-min intervals thereafter. The results are shown in Fig. 6. The control sample showed a very slight asymmetry on the leading edge, but was otherwise homogeneous and had an s20,w = 5.2s. In the presence of 0.5% sodium dodecyl sulfate there were two components: approximately 85% of a major component with s20,W= 2.8S, and a minor component
FIG.
+ 2000
- 200
415
420
42.5
43.0
43.5
440
445
X2 (cm21
418
STEVEKS,
FIG. G. Sedimentation-velocity in 0.035 M Verona1 + 0.065 M NaCl sulfate; bottom sample: same + min intervals starting 28 min after
BROWN,
AND
SMITH
ultracentrifugation. Top sample: 10 mg/ml lipoxygenase at pH 7.5; middle sample: same + 0.5% sodium dodecyl 1.0% sodium dodecyl sulfate. Pictures were taken at 8attaining speed.
(-15%) of S‘&,W = 6.3s. In 1% sodium dodecyl sulfate the concentration of the heavier minor component decreased to approximately 5 %. Under these conditions the szo,Wvalues were 2.35 and 5.7s. Amino acid composition. Table II presents the amino acid composition of purified lipoxygenase. Since analysis showed that there was less than 1% carbohydrate in the preparation, the amino acid composition ww calculated on the basis of ratios of amino acids and a molecular weight of 108,000. No unusual amino acids were obqyrved on the recorder charts of the analyzer. Absorption spectrum of lipoxygenase. Figure 7 shows the absorption spectrum of purified lipoxygenase in 0.1 M sodium phosphate buffer at pH 7.4 and also in 0.1 M NaOH. From the latter, using the absorption at 294 rnh and 280 rnp, the tyrosine/ tryptophan ratio was estimated to be 3.00. A sample of known absorbance at 280 rnp was hydrolyzed in 6 N HCl, and its protein
concentration was calculated from the amino acid analysis. The E:z& (280 rnp) was found to be 17.4. This value was used for calculating protein concentration in several experiments. As is evident from Fig. 7, the spectrum is that of a simple protein. The ratio of absorbance of 280-260 rnp is 2.3. There is no evidence for the presence of a prosthetic group. Determination of suljhydryl groups. With the Ellman reagent (21, 22) a sample of protein in 0.02 M sodium phosphate buffer at pH 8.0 gave complete color development in 90 min. Only 0.2 free sulfhydryl groups per molecule of protein could be detected. In the same buffer but containing 0.5% sodium dodecyl sulfate as denaturing agent, the color development was complete in less than 10 min and 3.92 sulfhydryl groups per molecule of protein were found. For determination of free sulfhydryl groups as carboxymethylcysteine, 10 mg of purified soybean lipoxygenase were dissolved
SOYBEAN TABLE AMINO
ACID
419
LIPOXYGENASE
II
COMPOSITION LIPOXYGENASE
OF SOYBEAK
Residues per molecule of enzyme Amino acid
Lysine Histidine Arginine Aspartic acid Threoninea Serinea Glutamic acid Proline Glycine Alanine Half-cystineb Valinec Methionine Isoleucinec Leucine TyrosinePhenylalanine Tryptophan Ammoniaa Total
Average or extrapolated value
53.85 27.23 42.19 110.17 56.20 74.70 102.97 59.56 68.67 72.42 7.69 64.90 16.98 59.63 102.20 40.80 37.35 13.6W 20.224 102.44
Residues to nearest integer
54 27 42 110 56 75 103 60 69 72 8 65 17 60 102 41 37 20 102 1018’
-Values calculated by linear extrapolation to zero hydrolysis time. *Value determined as cysteic acid after 24-hr hydrolysis of performic acid-oxidized lipoxygenase. ~Average value from 70-hr hydrolyzates only. dValue determined spectrophotometrically using tyrosine : tryptophan ratio. eValue determined by the method of Spies and Chambers (17). f Value for ammonia is omitted.
in 2 ml of 0.1 M sodium phosphate buffer at pH 7.5, 0.005 M in EDTA and 4 M in guanidine hydrochloride. To this solution was added a lOO-fold molar excess of recrystallized iodoacetic acid, and the sample was adjusted to pH 8.0 by addition of a concentrated NaOH solution. After 30 min at room temperature, the solution was dialyzed for 48 hr against five changes of 1 liter of deionized water containing Amberlite MB1 mixed-bed resin. To 1 ml of this solution there was added 1 ml of concentrated HCl. The sample was hydrolyzed in a sealed, evacuated tube at 110’ for 22 hr and the
300 250 WAVELENGTH
350
4 0
IN rn$
FIG. 7. Absorption spectrum of purified soybean lipoxygenase. The protein concentration was approximately 0.4 mg/ml and the samples were measured against a buffer blank. Solvent systems are as indicated in the figure.
amount of S-carboxymethyl cysteine present was determined by amino acid analysis. By this method 2.92 residues (uncorrected) per molecule of protein were found. Inasmuch as some destruction always occurs, this result is in reasonable accord with the four residues found by the Ellman method. DISCUSSION
Our purified lipoxygenase preparation is comparable in enzymic activity to those described by Theorell el al. (3) and by Mitsuda et al. (12). We attempted to repeat the last step of purification described by Mitsuda et al. (12) which consisted of absorbing the lipoxygenase on a column of CM-Sephadex G-50 equilibrated with 0.125 M acetate buffer. These investigators (12) reported that the enzyme is retained under these conditions but can be eluted by 0.175 M acetate buffer at pH 5.5. In our hands under these conditions, the active fraction remained bound to the column but could be eluted, without significant further purification, by increasing the salt concentration to 0.5 M NaCI.
420
STEVENS.
BROWN.
The value of 108,000 obtained for the molecular weight by sedimentation-equilibrium is in accord with that obtained by Theorell et al. (3), namely, 102,000 by sedimentation and diffusion, and with that found by Mitsuda et al. (la), 102,000 =t 3,000, by thin-layer gel filtration on Sephadex G-100 plates. In comparing our amino acid composition to the one reported by Holman et al. (5), which was obtained by paper chromatographic and microbiological techniques, there are a few differences that should be mentioned. Holman et al. (5) reported the presence of hydroxylysine and also of an unknown residue. Such residues could not be detected in our preparation. Holman et al. (5) were not able to detect cystine in their preparation and concluded that it is either absent or occurs in very low amounts. We find eight residues of half-cystine (lowest of any amino acid) per molecule of protein, estimated after oxidation to cysteic acid. Thus, there appear to be four residues of cysteine and four of half-cystine per molecule of lipoxygenase. Nevertheless, various thiol reagents do not inhibit lipoxygenase, as previously reported (1) and as confirmed by us. In our experiments, we found that four free sulfhydryl groups could be detected by the Ellman reagent in the presence of sodium dodecyl sulfate or guanidine hydrochloride, whereas only 0.2 residues per molecule reacted with the Ellman reagent when no denaturing agent was present. This indicates that in the native molecule the sulfhydryl groups are not available for reaction and may explain why thiol reagents do not inactivate the enzyme, although thiol groups may be involved in the mechanism of reaction. We have also confirmed earlier reports (1) that a large variety of metal-combining reagents have no inhibitory effect on the enzyme whatsoever. Dissociation of the lipoxygenase molecule into subunits was incomplete. After dialysis against 6 M guanidine hydrochloride containing 0.5 % mercaptoethanol, the mixture became heterogeneous as judged by sedimentation-equilibrium ultracentrifugation. At least three molecular species were pres-
AND
SMITH
ent: one with an apparent molecular weight of 55,000, presumably representing a subunit, one with a molecular weight of approximately 112,000, apparently the original molecule, and some heavier material which settled at the bottom of the cell and presumably consisted of aggregated material. In sedimentation experiments addition of 0.5% sodium dodecyl sulfate reduced the sedimentation coefficient of the sample from 5.2s to 2.8s. Taking into account the binding of sodium dodecyl sulfate to the protein, this new species presumably represents a subunit of half the molecular weight. The second minor component found in the ultracentrifuge pattern in the presence of 0.5% sodium dodecyl sulfate, with a sedimentation coefficient of 6.3 S, could then represent some undissociated material with bound sodium dodecyl sulfate. This is supported by the fact that raising the sodium dodecyl sulfate concentration to 1% results in a decrease of the minor component to a level in accordance with the slight asymmetry found on the leading edge of the ultracentrifuge pattern of the native material without sodium dodecyl sulfate. The amino acid composition of the protein shows no unusual features although, as noted, both sulfhydryl groups and cystine are present. Lipoxygenase still remains as an unusual example of an oxygenase which lacks a prosthetic group and requires no cofactor or metal ion for its activity. As noted above, there is no evidence that the masked sulfhydryl groups participate in the reaction. ACKNOWLEDGMENT We are indebted to Miss Dorothy McNall her help with the amino acid analyses.
for
REFERENCES 1. TAPPEL, A. L., in “The Enzymes” (Boyer, P. D., Lardy, H., and Myrbiick, K., eds.), Vol. 8, p. 275. Academic Press, New York (1963). 2. AND& E., AND Hou, K. W., C. R. dead. Sci. 194, 645 (1932). 3. THEORELL, H., HOLMAN, R.T., AND~ESON, A., Acta Chsm. &and. 1, 571 (1947). 4. HOLMAN, R. T., Arch. Biochem. Biophys. 16, 403 (1947).
SOYBEAN
LIPOXYGENASE
5. HOLMAN, R. T., PANZER, F., SCHWEIGERT, B. S., AND AMES, S. R., Arch. Biochem. Biophys. 26, 199 (1950). 6. TAPPEL, A. L., BOYER, P. D., AND LUNDBERG, W. O., J. Biol. Chem. 199, 267 (1952). 7. HAMBERG, M., AND SAMUELSSON, B., Biochem. Bioph.ys. Res. Commun. 21, 531 (1965). 8. WALKER, G. C., Biochem. Biophys. Res. Commun. 13, 431 (1963). 9. DOLEV, A., MOUNTS, T. L., ROHWEDDER, W. K., AND DUTTON, H. J., Lipids 1,293 (1966). 10. DOLEV, A., ROHWEDDER, W. K., AND DUTTON, H. J., Lipids 2,28 (1967). 11. DOLEV, A., ROHWEDDER, W. K., MOUNTS, T. L., AND DUTTON, H. J., Lipids 2,33 (1967). 12. MITSUDA, H., YASUMOTO, K., YAMAMOTO, A., AND KUSANO, T., Agr. Biol. Chem. 31, 115 (1967). 13. MITsTJDA, H., YASIJMOTO, K., AND YAMAMOTO, A., Arch. Biochem. Biophys. 118, 664 (1967). 14. ALLEN, J. C., European J. Biochem. 4, 201 (1968).
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15. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S., Anal. Chem. 30, 1190 (1958). 16. BEAVEN, G. H., AND HOLIDAY, E. R., Advan. Protein Chem. 7, 319 (1952). 17. SPIES, J. R., AND CHAMBERS, D. C., Anal. Chem. 21, 1249 (1949). 18. MOORE, S., J. Biol. Chem. 236, 235 (1963). 19. TSUGITA, A., AND AKABORI, S., J. Biochem. 46, 695 (1959). 20. CARSTEN, M. E., AND PIERCE, J. G., J. Biol. Chem. 238, 1724 (1963). 21. ELLMAN, G. L., Arch. Biochem. Biophys. 82, 70 (1959). 22. FERNANDEZ-DIEZ, M. J., OSUGA, D. T., AND FEENEY, R. E., Arch. Biochem. Biophys. 107, 449 (1964). 23. CRESTFIELD, A. M., MOORE, S., AND STEIN, W. H., J. Biol. Chem. 238,622 (1963). 24. ORNSTEIN, L., AND DAVIS, B. J., Disc Electrophoresis, Distillation Products Industries, Rochester, New York (1964). 25. YPHANTIS, D. H., Biochemistry 3,297 (1964).
OF
BIOCHEMISTRY
AND
Some FRITS Department
136, 413-421 (1970)
BIOPHYSICS
Properties
of Soybean
C. STEVENS?, DOUGLAS
o-f Biological
Chemistry, Received
UCLA
August
bipoxygenase’
M. BROWN,
School of Medicine, California 900.??4
11, 1969; accepted
AND EMIL University
November
L. SMITH3
of California,
Los ilngeles,
18, 1969
Commercial soybean lipoxygenase (EC 1.99.2.1) was purified by ammonium sulfate fractionation, gel filtration on Sephadex G-150, and chromatography on DEAEcellulose. The purified protein was essentially homogeneous as judged by acrylamide gel electrophoresis and ultracentrifugation. The molecular weight is 108,000 as determined by the sedimentation-equilibrium method. The amino acid composition was determined, and it was shown that the protein contains four residues of free sulfhydryl groups and four residues of half-cystine per molecule. Treatment of the protein with guanidine hydrochloride or sodium dodecyl sulfate produces dissociation. Present evidence indicates that the protein is composed of two subunits of 54,090 molecular weight.
Lipoxygenase (EC 1.99.2.1, linoleate:oxygen oxidoreductase) catalyzes the oxidation by moleculas oxygen of cis-methylene interrupted unsaturated fatty acids and their esters to respective hydroperoxides (1) : R-cH=CH--CH,-CH=CHR,
+ 0,
R-CH=CH-CH=CH-CH-R,
A
H09
The enzyme was discovered through its concurrent oxidation0 of carotene (2). Theorell, Holman, and Akeson (3) obtained a crystalline preparation from soybeans which was homogeneous, as judged by ultracentrifugation and moving boundary electrophoresis, and had an estimated molecular weight of 102,000 and an isoelectric point at pH 5.4. Holman (4) found that lipoxygenase 1 This investigation was aided by Grant GM 11061 from the National Institute of General Medical Sciences, United States Public Health Service. 2 Present address: Department of Biochemistry, Faculty of Medicine, University of Manitoba, Winnipeg 3, Manitoba, Canada. 3 To whom inquiries and requests for reprints should be addressed.
is not inhibited by pyrophosphate, fluoride, cyanide, azide, p-chloromercuribenzoate, mercury ions, or diethyldithiocarbamic acid, and concluded that the activity of the enzyme is not dependent on metal ions or sulfhydryl groups. Using the then available techniques of paper chromatography and microbiological assays, Holman et ~2. (5) also determined the amino acid composition of lipoxygenase. Tappel, Boyer, and Lundberg (6) studied the reaction mechanism of the enzyme and on the basis of their results proposed a simple mechanism involving formation of a biradical from linoleate and oxygen on the surface of the enzyme. The biradical may accept electrons from antioxidants or may react to give the conjugated linoleate peroxide. The extreme specificity of lipoxygenase was recently demonstrated by Hamberg and Samuelson (7). Walker (8) demonstrated by EPR spectroscopy the existence of free radicals during the course of linoleate peroxidation by the enzyme. Dolev and co-workers (9-11) showed in labeling experiments that the oxygen incorporated into the hydroperoxide product is derived from molecular oxygen and not from water.
414
STEVENS,
BROWN,
It should be noted that many of the studies on lipoxygenase have been carried out with commercial preparations, which were found to be heterogeneous in the present investigation. While our work was in progress Rfitsuda et al. (12) reported studies on the purified enzyme and investigated the effect of saturated monohydroalcohols on the reaction in order to elucidate the role of hydrophobic regions of enzyme on the binding of the substrate (13). Allen (14) has also reported an improved method for preparation of the enzyme. No attempts have been made recently to characterize the enzyme as a protein by modern methods, and the nature of the catalytic site is still not understood. We undertook the present study in order to obtain information concerning the subunit of this unstructure and the composition usual enzyme. EXPERIMENTAL
PROCEDURE
The starting material was a commercial preparation (Lipoxidase Lx 6EA) of soybean lipoxygenase obtained from the Worthington Biochemical Corporation, Freehold, New Jersey. A preparation (No. 389093) purchased from Fluka, A. G., Buchs, S. C., Switzerland, was also investigated. Chemicals. Sephadex G-150 (40-120 p) was obtained from Pharmacia Fine Chemicals, Inc., Nutley, New Jersey and DEAE-cellulose with an exchange capacity of 0.75 meq/g (Cellex-D No. 4551) was purchased from Bio-Rad Laboratories, Richmond, California. Reagent grade iodoacetic acid was recrystallized from petroleum ether (60110”). Guanidine hydrochloride was reagent grade (J. T. Baker Chemical Co.) and was washed with acetone to remove yellow color. The Ellman reagent, 5,5’-dithiobis-(2.nitro-benzoic acid) was obtained from the Aldrich Chemical Company, Inc., Milwaukee, Wisconsin, and sodium dodecylsulfate was a commercial preparation (Sipon SD) of the American Acoolac Corporation, Baltimore, Maryland. All other chemicals used were reagent grade. Enzyme assays. Lipoxygenase activity was determined by the following method. One hundred microliters of chromatographically pure linoleic acid (Calbiochem) were dissolved in 15 ml of absolute ethyl alcohol, and water was added to make 25 ml of stock solution; this solution was made fresh every day. Immediately before use, 5 mi of substrate stock solution were diluted with 25 ml of 0.2 M sodium borate buffer at pH 9.0, and
AND
SMITH
the solution was oxygenated by bubbling gaseous oxygen through for a few minutes. The enzyme solution to be assayed was also diluted with 0.2 M sodium borate buffer at pH 9.0 to give an absorbance reading of 0.050 at 280 rnh. Two milliliters of the diluted substrate solution were pipetted into a I-cm silica glass cuvette, and at zero time 0.05-l ml of enzyme solution and buffer were added to make a total volume of 3 ml. The change in absorbance at 234 mp, produced by formation of hydroperoxide from the substrate, was measured as a function of time in a Zeiss M4QIII spectrophotometer. Readings were taken every 30 set for several minutes. One enzyme unit is defined as a change in absorbance (A) of 0.01 in 1 min. Specific activity of enzyme preparations are expressed as number of enzyme units per 0.01 absorbance unit at 280 rnM of the enzyme solution (Am). With very dilute enzyme solutions there was an initial lag period and the change in absorption at 234 mp became linear only after 1 min. All measurements were taken in the linear portion of the change in absorbance with time. Under these conditions enzyme activity was strictly proportional to protein concentration. Amino acid analyses. Samples of the purified protein were thoroughly dialyzed, first against 0.1 M KC1 and then against deionized water. After lyophilization, aliquots were hydrolyzed in threetimes glass-distilled 6 N HCl at 110” in sealed, evacuated tubes. Analyses were performed in duplicate after 24 and 70 hr of hydrolysis on the Spinco automatic amino acid analyzer by the method of Spackman, Stein, and Moore (15). Tryptophan was determined spectrophotometrically (16) and also by the calorimetric method of Spies and Chambers (17). The half-cystine content was determined after performic acid oxidation by the method of Moore (18). Total hexose was determined by the method of Tsugita and Akabori (19), as modified by Carsten and Pierce (20). Free sulfhydryl groups were determined by the method of Ellman (21)) as described by FernandezDiez, Osuga, and Feeney (22). Alternatively, sulfhydryl groups were estimated as S-carboxymethylcysteine by amino acid analysis after alkylation by the method of Crestfield, Moore, and Stein (23). Ultracentrifuge studies. Equilibrium experiments were performed with a Spinco Model E ultracentrifuge equipped with interference optics and constant temperature control (RTIC). Sedimentation velocity studies were run at 59,780 rpm Acrylamide gel electrophoresis. Acrylamide gel electrophoresis was performed in 7.5% gels in Tris-glycine buffer at pH 8.8, as described by
SOYBEAN
LIPOXYGENASE
Ornstein and Davis (24). Samples were applied to the top of the gel in buffer containing 15y0 sucrose.
415 TABLE
PURIFICATION
OF
SOYBEAN
I LIPOXYGENASE
RESULTS
Purijication. Figure 1 shows the acrylamide electrophoresis patterns of preparations of lipoxygenase obtained from Worthington and Fluka, respectively. Both preparations were heterogeneous by this technique. Sedimentation velocity centrifugation of Worthington lipoxygenase at pH 7.4 (0.035 M Verona1 + 0.065 M NaCl) revealed at least three overlapping peaks. Both commercial preparations had an identical specific activity of 49 enzyme units per absorbance unit. The Worthington preparation was purified as described below; results are summarized in Table I. Figure 2 shows the electrophoretic pattern in acrylamide gel at various stages of purification. A six-fold purification was achieved with a 50 to 60 % recovery of the original activity.
FIG. 1. Acrylamide gel electrophoresis of commercial lipoxygenase: left, 250 pg of Worthington lipoxygenase; right, 250 rg of Fluka lipoxygenase. The samples were run on 7.57” gels at a constant current of 5 mA per tube for 50 min in the cold. They were stained with amido schwarz.
Crude lipoxygenase (Worthington) (NH&SOa fractionation active fraction on 40-60% cut Gel filtration on Sephadex G-150 60% saturated (NH&S04 precipitate of active peak Chromatography on DEAE-cellulose 60% saturated (NH&SO4 precipitate of active peak
49
100
65
95
130
82
300
60
a The activity was measured as described in the Experimental section.
a. Ammonium sulfate fractionation. Lipoxygenase (8 g) was dissolved in 400 ml of 30% saturated ammonium sulfate at 4O and then brought to 40% saturation by addition of solid ammonium sulfate. After standing overnight at 4” the inactive precipitate was removed by centrifugation and discarded. The supernatant solution was brought to 60 % saturation by addition of solid ammonium sulfate and after standing overnight in the cold, the precipitate was collected by centrifugation. This fraction, accounting for almost all of the original activity and for approximately 80% of the original protein, was redissolved in 50 ml of 0.1 31 ammonium bicarbonate and dialyzed against three changes of 4 liters of the same buffer over a period of 48 hr. This solution was stable and showed no loss of activity over a period of more than a month in the cold. The treatment increased the specific activity of the enzyme from 49-65 enzyme units/absorbance unit. b. GelJiltration on Xephadex G-150. Approxmately 20-ml batches of the lipoxygenase solution in 0.1 M (NH,)HCO;( were applied at 4O to a 3.8 X 127-cm column of Sephadex G-150 (40-120 cl) equilibrated with the same buffer. Elution was performed with the same buffer under a hydrostatic pressure of approximately lo-20 cm of water. This gave a flow rate of 40-50 ml/hr, and 10 tubes per hour were collected with the
416
STEVENS,
BROWN, AND SMITH
TUBE
NUMBER
FIG. 3. Purification of ammonium sulfate-fractionated Worthington lipoxygenase on Sephadex G-150. The details are described in the text. The absorbance at 280 mc: (0) and the enzymatic activity (x) were determined on effluent fractions. The solid bar indicates the active fractions pooled.
FIG. 2. Acrylamide gel electrophoresis of lipoxygenase at various stages of purification. From left to right: 250 pg of Worthington lipoxygenase, 100 rg of the act,ive fraction after gel filtration on Sephadex G-150, 50 fig of the active fraction after chromatography on DEAE-cellulose, 100 fig of the active fraction after chromat,ography on DEAE-cellulose. The samples were run on 7.5y0 gels at a constant current of 4 mA per tube in the cold and stained with amido schwarz.
aid of an automatic fraction collector. l’rotein absorption at 280 rnp and enzymic activity were determined on alternate fractions. Figure 3 shows a typical elution pattern. Active fractions from consecutive runs on the same column were pooled and the protein was precipitated by bringing the solution to 60% saturation by addition of solid ammonium sulfate at 4’. This material had a specific activity of 130 enzyme units/ absorbance unit. Acrylamide gel electrophoresis (Fig. 2) showed that there were
two major and some minor components still present. c. Column chromatography on DEAE-cellulose. Further purification was achieved by chromatography on DEAE-cellulose at 4’ with a linear gradient from 0.02 M sodium phosphate at pH 7.5 to 0.02 M sodium phosphate at pH 7.5 + 0.5 M NaCl. The details of the run and a typical elution pattern are shown in Fig. 4. Since the enzyme was rather unstable under these conditions, the active fractions were pooled, as soon as they were located, and solid ammonium sulfate was added to 60% saturation. The purified enzyme could be kept at 4’ indefinitely under these conditions without noticeable loss of activity; however, it loses activity upon dialysis and lyophilization. The purified lipoxygenase had a specific activity of 300 units per absorbance unit and gave a single band on acrylamide gel electrophoresis at pH 8.8 (Fig. 2). Molecular weight determination. The molecular weight of purified lipoxygenase was determined by the high-speed sedimentation-equilibrium method of Yphantis (25). The partial specific volume (V) of the protein was calculated from its amino acid composition (see below) as 0.734. A sample of lipoxygenase was dialyzed overnight against 0.035 M sodium Verona1 buffer f 0.065 M NaCl at pH 7.4. A three-
SOYBEAN
TUBE
LIPOXYGENASE
NUMBER
4. Purification of lipoxygenase on DEAEcellulose. Approximately 160 mg of the active Sephadex G-150 fraction were dissolved in and dialyzed overnight against 0.02 M sodium phosphate buffer at pH 7.5. The sample (3 ml) was applied to a column of DEAE-cellulose (1.7 X 15 cm) equilibrated with the same buffer. A flow rate of 36 ml/hr was maintained with a Beckman Accuflow pump and 4-ml fractions were collected. Separation was achieved by passing through 100 ml of 0.02 M sodium phosphate buffer at pH 7.5, followed by a linear gradient which was established with 100 ml 0.02 M sodium phosphate buffer, pH 7.5 + 0.5 M NaCl.
FIG. 5. Sedimentation-equilibrium ultracentrifugation of soybean lipoxygenase. Plot of logarithm of the concentration (C) in fringes versus radial distance squared is shown at 22 hr after reaching a speed of 14,000 rpm. Concentration shown is 0.6 mg/ml .
channel equilibrium cell was filled with 0.4, 0.6, and 1.0 mg/ml protein solution and run at 14,000 rpm after overspeeding for 1 hr at 20,000 rpm. The temperature was maintained at 20’ throughout the run. The plates were read at each half fringe on the Gaertner microcomparator at 22 hr after attaining equilibrium speed. The calculations were programmed into a Monroe Epic 3000 computer. The results for the middle channel are shown in Fig. 5. The sample was found to contain two molecular components with the molecular weight at the meniscus M, = 108,000 f 2,000. The small amount (approximately 5 % from sedimentation-velocity determination reported below) of heavier material had a molecular weight greater than 188,000 and could represent some dimerization. Subunit structure of soybean lipoxygenase. For a sedimentation-equilibrium experiment a sample was dialyzed for 48 hr against 6 M guanidine hydrochloride containing 0.5 % mercaptoethanol at pH 7.7. Concentrations of 0.4, 0.6, and 0.8 mg/ml were placed into the three-ch.annel Yphantis-type cell, and
the run was performed for 20 hr at 24,630 rpm. The material was found to be heterogeneous, giving at the meniscus M, = 58,000 and at the bottom of the cell Mb = 112,000. There was also a small amount of unresolved heavier material at the bottom of the cell. It is evident that only partial dissociation of the protein wa.s obtained in this experiment. By the technique of sedimentation velocity, three samples were studied at a concentration of 10 mg/ml: (1) in 0.035 31 Verona1 + 0.64 M NaCl at pH 7.4; (2) in the same buffer + 0.5% sodium dodecyl sulfate; (3) in the same buffer + 1% sodium dodecyl sulfate. These samples were run at 59,780 rpm at 20”. Pictures were taken in all three cases starting at 28 min after attaining speed and then at 8-min intervals thereafter. The results are shown in Fig. 6. The control sample showed a very slight asymmetry on the leading edge, but was otherwise homogeneous and had an s20,w = 5.2s. In the presence of 0.5% sodium dodecyl sulfate there were two components: approximately 85% of a major component with s20,W= 2.8S, and a minor component
FIG.
+ 2000
- 200
415
420
42.5
43.0
43.5
440
445
X2 (cm21
418
STEVEKS,
FIG. G. Sedimentation-velocity in 0.035 M Verona1 + 0.065 M NaCl sulfate; bottom sample: same + min intervals starting 28 min after
BROWN,
AND
SMITH
ultracentrifugation. Top sample: 10 mg/ml lipoxygenase at pH 7.5; middle sample: same + 0.5% sodium dodecyl 1.0% sodium dodecyl sulfate. Pictures were taken at 8attaining speed.
(-15%) of S‘&,W = 6.3s. In 1% sodium dodecyl sulfate the concentration of the heavier minor component decreased to approximately 5 %. Under these conditions the szo,Wvalues were 2.35 and 5.7s. Amino acid composition. Table II presents the amino acid composition of purified lipoxygenase. Since analysis showed that there was less than 1% carbohydrate in the preparation, the amino acid composition ww calculated on the basis of ratios of amino acids and a molecular weight of 108,000. No unusual amino acids were obqyrved on the recorder charts of the analyzer. Absorption spectrum of lipoxygenase. Figure 7 shows the absorption spectrum of purified lipoxygenase in 0.1 M sodium phosphate buffer at pH 7.4 and also in 0.1 M NaOH. From the latter, using the absorption at 294 rnh and 280 rnp, the tyrosine/ tryptophan ratio was estimated to be 3.00. A sample of known absorbance at 280 rnp was hydrolyzed in 6 N HCl, and its protein
concentration was calculated from the amino acid analysis. The E:z& (280 rnp) was found to be 17.4. This value was used for calculating protein concentration in several experiments. As is evident from Fig. 7, the spectrum is that of a simple protein. The ratio of absorbance of 280-260 rnp is 2.3. There is no evidence for the presence of a prosthetic group. Determination of suljhydryl groups. With the Ellman reagent (21, 22) a sample of protein in 0.02 M sodium phosphate buffer at pH 8.0 gave complete color development in 90 min. Only 0.2 free sulfhydryl groups per molecule of protein could be detected. In the same buffer but containing 0.5% sodium dodecyl sulfate as denaturing agent, the color development was complete in less than 10 min and 3.92 sulfhydryl groups per molecule of protein were found. For determination of free sulfhydryl groups as carboxymethylcysteine, 10 mg of purified soybean lipoxygenase were dissolved
SOYBEAN TABLE AMINO
ACID
419
LIPOXYGENASE
II
COMPOSITION LIPOXYGENASE
OF SOYBEAK
Residues per molecule of enzyme Amino acid
Lysine Histidine Arginine Aspartic acid Threoninea Serinea Glutamic acid Proline Glycine Alanine Half-cystineb Valinec Methionine Isoleucinec Leucine TyrosinePhenylalanine Tryptophan Ammoniaa Total
Average or extrapolated value
53.85 27.23 42.19 110.17 56.20 74.70 102.97 59.56 68.67 72.42 7.69 64.90 16.98 59.63 102.20 40.80 37.35 13.6W 20.224 102.44
Residues to nearest integer
54 27 42 110 56 75 103 60 69 72 8 65 17 60 102 41 37 20 102 1018’
-Values calculated by linear extrapolation to zero hydrolysis time. *Value determined as cysteic acid after 24-hr hydrolysis of performic acid-oxidized lipoxygenase. ~Average value from 70-hr hydrolyzates only. dValue determined spectrophotometrically using tyrosine : tryptophan ratio. eValue determined by the method of Spies and Chambers (17). f Value for ammonia is omitted.
in 2 ml of 0.1 M sodium phosphate buffer at pH 7.5, 0.005 M in EDTA and 4 M in guanidine hydrochloride. To this solution was added a lOO-fold molar excess of recrystallized iodoacetic acid, and the sample was adjusted to pH 8.0 by addition of a concentrated NaOH solution. After 30 min at room temperature, the solution was dialyzed for 48 hr against five changes of 1 liter of deionized water containing Amberlite MB1 mixed-bed resin. To 1 ml of this solution there was added 1 ml of concentrated HCl. The sample was hydrolyzed in a sealed, evacuated tube at 110’ for 22 hr and the
300 250 WAVELENGTH
350
4 0
IN rn$
FIG. 7. Absorption spectrum of purified soybean lipoxygenase. The protein concentration was approximately 0.4 mg/ml and the samples were measured against a buffer blank. Solvent systems are as indicated in the figure.
amount of S-carboxymethyl cysteine present was determined by amino acid analysis. By this method 2.92 residues (uncorrected) per molecule of protein were found. Inasmuch as some destruction always occurs, this result is in reasonable accord with the four residues found by the Ellman method. DISCUSSION
Our purified lipoxygenase preparation is comparable in enzymic activity to those described by Theorell el al. (3) and by Mitsuda et al. (12). We attempted to repeat the last step of purification described by Mitsuda et al. (12) which consisted of absorbing the lipoxygenase on a column of CM-Sephadex G-50 equilibrated with 0.125 M acetate buffer. These investigators (12) reported that the enzyme is retained under these conditions but can be eluted by 0.175 M acetate buffer at pH 5.5. In our hands under these conditions, the active fraction remained bound to the column but could be eluted, without significant further purification, by increasing the salt concentration to 0.5 M NaCI.
420
STEVENS.
BROWN.
The value of 108,000 obtained for the molecular weight by sedimentation-equilibrium is in accord with that obtained by Theorell et al. (3), namely, 102,000 by sedimentation and diffusion, and with that found by Mitsuda et al. (la), 102,000 =t 3,000, by thin-layer gel filtration on Sephadex G-100 plates. In comparing our amino acid composition to the one reported by Holman et al. (5), which was obtained by paper chromatographic and microbiological techniques, there are a few differences that should be mentioned. Holman et al. (5) reported the presence of hydroxylysine and also of an unknown residue. Such residues could not be detected in our preparation. Holman et al. (5) were not able to detect cystine in their preparation and concluded that it is either absent or occurs in very low amounts. We find eight residues of half-cystine (lowest of any amino acid) per molecule of protein, estimated after oxidation to cysteic acid. Thus, there appear to be four residues of cysteine and four of half-cystine per molecule of lipoxygenase. Nevertheless, various thiol reagents do not inhibit lipoxygenase, as previously reported (1) and as confirmed by us. In our experiments, we found that four free sulfhydryl groups could be detected by the Ellman reagent in the presence of sodium dodecyl sulfate or guanidine hydrochloride, whereas only 0.2 residues per molecule reacted with the Ellman reagent when no denaturing agent was present. This indicates that in the native molecule the sulfhydryl groups are not available for reaction and may explain why thiol reagents do not inactivate the enzyme, although thiol groups may be involved in the mechanism of reaction. We have also confirmed earlier reports (1) that a large variety of metal-combining reagents have no inhibitory effect on the enzyme whatsoever. Dissociation of the lipoxygenase molecule into subunits was incomplete. After dialysis against 6 M guanidine hydrochloride containing 0.5 % mercaptoethanol, the mixture became heterogeneous as judged by sedimentation-equilibrium ultracentrifugation. At least three molecular species were pres-
AND
SMITH
ent: one with an apparent molecular weight of 55,000, presumably representing a subunit, one with a molecular weight of approximately 112,000, apparently the original molecule, and some heavier material which settled at the bottom of the cell and presumably consisted of aggregated material. In sedimentation experiments addition of 0.5% sodium dodecyl sulfate reduced the sedimentation coefficient of the sample from 5.2s to 2.8s. Taking into account the binding of sodium dodecyl sulfate to the protein, this new species presumably represents a subunit of half the molecular weight. The second minor component found in the ultracentrifuge pattern in the presence of 0.5% sodium dodecyl sulfate, with a sedimentation coefficient of 6.3 S, could then represent some undissociated material with bound sodium dodecyl sulfate. This is supported by the fact that raising the sodium dodecyl sulfate concentration to 1% results in a decrease of the minor component to a level in accordance with the slight asymmetry found on the leading edge of the ultracentrifuge pattern of the native material without sodium dodecyl sulfate. The amino acid composition of the protein shows no unusual features although, as noted, both sulfhydryl groups and cystine are present. Lipoxygenase still remains as an unusual example of an oxygenase which lacks a prosthetic group and requires no cofactor or metal ion for its activity. As noted above, there is no evidence that the masked sulfhydryl groups participate in the reaction. ACKNOWLEDGMENT We are indebted to Miss Dorothy McNall her help with the amino acid analyses.
for
REFERENCES 1. TAPPEL, A. L., in “The Enzymes” (Boyer, P. D., Lardy, H., and Myrbiick, K., eds.), Vol. 8, p. 275. Academic Press, New York (1963). 2. AND& E., AND Hou, K. W., C. R. dead. Sci. 194, 645 (1932). 3. THEORELL, H., HOLMAN, R.T., AND~ESON, A., Acta Chsm. &and. 1, 571 (1947). 4. HOLMAN, R. T., Arch. Biochem. Biophys. 16, 403 (1947).
SOYBEAN
LIPOXYGENASE
5. HOLMAN, R. T., PANZER, F., SCHWEIGERT, B. S., AND AMES, S. R., Arch. Biochem. Biophys. 26, 199 (1950). 6. TAPPEL, A. L., BOYER, P. D., AND LUNDBERG, W. O., J. Biol. Chem. 199, 267 (1952). 7. HAMBERG, M., AND SAMUELSSON, B., Biochem. Bioph.ys. Res. Commun. 21, 531 (1965). 8. WALKER, G. C., Biochem. Biophys. Res. Commun. 13, 431 (1963). 9. DOLEV, A., MOUNTS, T. L., ROHWEDDER, W. K., AND DUTTON, H. J., Lipids 1,293 (1966). 10. DOLEV, A., ROHWEDDER, W. K., AND DUTTON, H. J., Lipids 2,28 (1967). 11. DOLEV, A., ROHWEDDER, W. K., MOUNTS, T. L., AND DUTTON, H. J., Lipids 2,33 (1967). 12. MITSUDA, H., YASUMOTO, K., YAMAMOTO, A., AND KUSANO, T., Agr. Biol. Chem. 31, 115 (1967). 13. MITsTJDA, H., YASIJMOTO, K., AND YAMAMOTO, A., Arch. Biochem. Biophys. 118, 664 (1967). 14. ALLEN, J. C., European J. Biochem. 4, 201 (1968).
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15. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S., Anal. Chem. 30, 1190 (1958). 16. BEAVEN, G. H., AND HOLIDAY, E. R., Advan. Protein Chem. 7, 319 (1952). 17. SPIES, J. R., AND CHAMBERS, D. C., Anal. Chem. 21, 1249 (1949). 18. MOORE, S., J. Biol. Chem. 236, 235 (1963). 19. TSUGITA, A., AND AKABORI, S., J. Biochem. 46, 695 (1959). 20. CARSTEN, M. E., AND PIERCE, J. G., J. Biol. Chem. 238, 1724 (1963). 21. ELLMAN, G. L., Arch. Biochem. Biophys. 82, 70 (1959). 22. FERNANDEZ-DIEZ, M. J., OSUGA, D. T., AND FEENEY, R. E., Arch. Biochem. Biophys. 107, 449 (1964). 23. CRESTFIELD, A. M., MOORE, S., AND STEIN, W. H., J. Biol. Chem. 238,622 (1963). 24. ORNSTEIN, L., AND DAVIS, B. J., Disc Electrophoresis, Distillation Products Industries, Rochester, New York (1964). 25. YPHANTIS, D. H., Biochemistry 3,297 (1964).