Substitution Reactions of Linoleic Acid ... - PubAg - USDA

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ciS-9-octadecenoic acid; B2, 13-ethylthio-12-oxo-cis-9- octadecenoic ... octadecenoic acid; and B, 13-linoleoyloxy-12-oxo-cis- ... Hydroxyoctadecadienoic. 66.7.
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LIPIDS,

Vol. 10, No.8, Pages: 448-453 (19i5)

Substitution Reactions of Linoleic Acid Hydroperoxide Isomerase 1 D.O. CHRISTIANSON and H.W. GARDNER, Northern Regional Research LaboratorY,2 Peoria, Illinois 61604

nates from elsewhere, presumably from a solvent. Gardner (3) isolated three products from the isomerization of linoleic acid hydroperoxide in the presence of linoleic acid and corn germ isomerase. The product fatty acids were 9hy droxy-l 0-oxo-cis-12-octadecenoic acid (aketol), 13-hYdroxy-10-oxo-trans-ll-octadecenoic acid (i-ketol) , and the linoleoyloxy ester of the a-ketol, 9-(cis-9,cis-12-octadecadie noylo xy)-l 0-oxo-cis-12-octadecenoic acid (B). He postulated that acylation occurred during formation of B and was catalyzed by another enzyme; however, the involvement of an acylase was not proved. Gardner also showed that B would not form when the a-ketol was incubated with linoleic acid in the presence of the corn germ enzyme preparation, but that formation of B only occurred when linoleic acid hydroperoxide and linoleic acid were incubated with the enzyme. Upon further examination of the enzyme reaction, we found that other fatty acids and other nucleophiles will substitute for the hydroperoxy group in the presence of corn germ isomerase. We also describe a wheat germ isomerase that in the presence of linoleic acid produces the same products as those obtained with corn germ isomerase.

ABSTRACT

Linoleic acid hydroperoxide isomerase was extracted from corn germ and partially purified by differential centrifugation. This enzyme catalyzed the isomerization of linoleic acid hydroperoxide (RCHOOH-CHtranFH-CH cis CH-R 1 ) to the expected a-ketol (R-eHOH-CO-CHzCH~H-R1) and i-ketol (R-CH 2 -CO-

CHtransCH-eH OH-R 1)' Isomerase also catalyzed the substitution of various reagents at the carbon bearing the hydroperoxide group. These fatty acid products had the following functional groupings: R-CHX-CO-CH 2 -CH cis CH-R 1 where X is either oleoyloxy, ethylthio, or methoxy resulting from the presence of oleic acid, ethanethiol, or methanol, respectively. A crude wheat germ extract containing both lipoxygenase and isomerase enzymes reacted with linoleic acid to yield a-ketols, i-ketols, and a sUbstitution product, the linoleoyloxy ester of a-ketol. Characterization of these products from wheat germ enzymes showed that the substitution reaction was not unique to corn germ. Because anions of the reagents tested are typical nucleophiles, the substit uti 0 n reactions may procee d by a nucleophilic mechanism as mediated by the isomerase enzyme.

EXPERIMENTAL PROCEDURES

Materials

INTRODUCTION

Zimmerman (l) has shown that flaxseed contains an enzyme that catalyzes the isomerization of linoleic acid hydroperoxides to monounsaturated a-ketols. When 1sO-labeled 1 3 -hydro peroxy -cis-9 ,tra ns-ll-octadecadienoic acid was the substrate for this flaxseed enzyme, only one oxygen atom of the l3-hydroperoxy function transfers to the 12-oxo group, the other is not retained in the product (2). This transfer indicates that a cyclic intermediate, cyclic peroxide, or epiperoxide is involved in the reaction and that the hydroxyl group origi-

(B37TMS X H84) (Oh43RF X A6l9) hybrid corn, Zea mays, came from the University of Illinois, Urbana. After harvest, the corn was dried with ambient air and stored at 0 F. Germ was prepared by a laboratory dry-milling operation. Germ dry milled from a soft red winter Wheat, Triticum aestivum, was supplied by Mennel Milling Co., Fostoria, Ohio. Lipoxygenase (EC 1.13.1.13) was obtained from Sigma Chemical Corp., St. Louis, Mo., (activity = 130,000 units/mg). The Hormel Institute, Austin, Minn., supplied linoleic acid (purity>99%). Oleic acid wwas purchased from Applied Science Laboratories, State College, Pa. Hydroperoxide Isomerase

1Presented at the AOeS Spring Meeting, Dallas, April 1975. 2ARS, USDA.

Isomerase was isolated by stirring hexanedefatted corn germ flour for 15 min in 0.1 M

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Official Use

LINOLEIC HYDROPEROXIDE ISOMERASE

phosphate buffer (l g germ/10m!) and by centrifuging at 8000 x g for 20 min to remove cellular debris. Further isomerase purification was achieved by recentrifuging the clarified supernatant at 78,400 x g for 1 hr to collect the particulate matter containing isomerase. The pellet, when resuspended in 0.2 M phosphate buffer at pH 6.9, served as the enzyme source for isomerization and substitution reactions. Specific activity of the resuspended pellet in 50 ml buffer (equivalent to 4 g defatted corn germ) was 15.6 pmoles/min 'mg protein. Isomerase activity was measured by the initial rate of decrease in conjugated diene absorption at 234 nm (3). The isomerase enzyme could be stored at -20 C in buffer for several days without substantial loss of activity. Protein content of the enzyme preparation was determined by the Folin-Wu procedure (4). In the wheat germ study, linoleic acid was oJddiz"d with an extract prepared from wheat germ. Full fat wheat germ (15 g) was extracted with 150 ml 0.1 M phosphate buffer (pH 6.9) and centrifuged at 10,000 x g for 20 min. The supernatant (l00 ml) was mixed directly with 500 pliter linoleic acid (K salt) and 500 pliter Tween in 70 ml water under bubbling oxygen for 50 min. Reaction was stopped by acidification to pH 4 with 1 N HCl. The products were extracted with CHCl r CH 3 0H 2: 1 (v/v) and separated by silicic acid column chromatography (3) with an elution gradient similar to that used for product separation obtained from corn germ sequential reaction.

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Methanol reaction contained 353 mg hydroperoxide (3.7 mM), 0.175 ml Tween, 61.2 ml methanol (20% by volume) and 250 pmoles/ min isomerase activity (equivalent to 10 g defatted germ) in a total volume of 301 ml. The product mixture was acidified to pH 4 with 1 N HCl and extracted with CHCI 3 CH 3 0H, 2: 1 (v/v). The organic phase containing fatty acid products was washed twice with water. The product mix was stored in ether at -20 C. Chromatography

Fatty acid products from the substitution reactions were separated by silicic acid column chromatography essentially under conditions described previously (3). Compounds were applied to the column in a slurry of 2 g silicic acid in hexane and eluted with a modified step-wise gradient; 350 ml 10% ether, 350 ml 20% ether, 350 ml 30% ether, 200 ml 40% ether, and 300 ml 50% ether in hexane. Fractions were monitored by Silica Gel G thin layer chromat ography (TLC) using isooctane-ether-acetic acid, 70:30: 1 (v/v/v) solvent system (double development). Products were detected on plates by spraying with 0.4% 2,4-dinitrophenylhydrazine in 2 N HCl or by charring with 50% H 2 S0 4 . Separated compounds were combined, evaporated to dryness under nitrogen, and stored in ether at -20 C for spectral analysis. For certain spectral analyses, the methyl ester of the products were prepared with diazomethane (6). Structural Characterization of Products

Substitution Reactions

Reactions were conducted with columnisolated hydroperoxide prepared by reaction of oxygen with linoleic acid in the presence of soy lipoxygenase. The method of oxidation, including the concentration of enzyme (units/ml), and the isolation of hydroperoxide were the same as used previously (5). The hydroperoxide was taken up in water in the presence of Tween, the reagent added and an equal volume of isomerase in 0.2 M phosphate buffer mixed for 1 hr. Oleic acid, ethanethiol, and methanol were the reagents used for the corn germ isomerase reactions. The oleic acid reaction mixture contained 213 mg hydro peroxide (3.6 mM), 0.927 ml Tween, 1.854 g oleic acid (0.034 M) and 150 pmoles/min isomerase activity (equivalent to 6 g defatted germ) in a total volume of 188 ml. The ethanethiol reaction contained 213 mg hydroperoxide (3.4 mM), 0.2 ml Tween, 2 ml ethanethiol (0.14 M) and 150 pmoles/min isomerase activity (equivalent to 6 g defatted germ) in a total volume of 198 ml.

NMR, IR, and mass spectra (MS) were used to characterize the structures of the products by methods described previously (7), with one exception: the MS of 13-o1eoyloxy-12-oxo-cis9-octadecenoic acid (methyl ester) was obtained from a probe sample rather than by gas liquid chromatography (GLC) elution. RESULTS

Identification of Substitution Products

Corn germ hydroperoxide isomerase action on substrate hydroperoxides in the presence of various reagents gave the usual products (a:- and ,-ketols) plus products resulting from substitution of the reagent anion with the hydroperoxy group. One was a fatty acid with the following functional moiety = R-CHX-COCH 2 -CH cis CH-R 1 where X is the anion of the reagent added. Since soybean lipoxygenase was used to produce the hydroperoxide, derivatives would be predominantly from 13-hydroperoxytrans-ll,cis-9-octadecadienoic acid (79%). One LIPIDS. VOL. 10. NO.8

D.O. CHRISTIANSON AND H.W. GARDNER

450

in Experimental Procedures were in ranges of 740-820 ml tor B 1 540-560 ml for B2 and 480-550 ml for B3 . For the purposes of this paper, we need not discuss the minor isomer of these three compounds. The substitution products were identified by their spectral properties as outlined. Product B 1: Assignment of resonances in NMR spectrum (Fig. 1) are as follows: the methoxyl protons absorb at 0 3.34 (S, 3H). The doublet at 3.248 is the methylene between the double bond and the oxo-group. Double irradiation of the olefinic proton centered at 0 5.54 (m, 2H) decoupled the absorption at 3.240 and thus confirmed the position of the olefin as Cl: to the C-ll methylene. Characteristic IR absorptions (B 1 methyi ester) were 1100 em-I, methoxy CoO stretch; 1720 em-I, keto carbonyl; and 1740 em-I, ester carbonyl. MS of product B 1 clearly supported the proposed structure. Replicate MS taken over the GLC peak showed mle 115 ion with greatest intensity (CH 3-[CH 2 ] 4-f~CH3) and mle

o

!'i\

~O B

0

'.0

OH

FIG. 1. NMR spectra of B I , 13-methow-12-oxociS-9-octadecenoic acid; B2 , 13-ethylthio-12-oxo-cis-9octadecenoic acid; B3 , 13-o1eoyloxy-12-oxo-cis-9octadecenoic acid; and B, 13-linoleoyloxy-12-oxo-cis9-octadecenoic acid.

115-31. Both M and M - 31 were absent. Product B 2: Assignments of resonances in the NMR spectrum of product B2 (Fig. 1) are as follows: methyl protons of ethylthio at o 1.16 (t, 3H); methylene protons of ethylthio centered around 0 2.38. Irradiation of the absorption at 0 1.16 confirmed the position of the methyl Cl: to the methylene of the ethylthio group. Irradiation of the olefinic protons centered at 0 5.52 (m, 2H) confirmed its position Cl: to the C-ll methylene. Characteristic IR absorptions (B 2 methyl ester) were 1720 em-I, keto carbonyl; 1740 em-I, ester carbonyl; and 730 em-I, sulfide absorption (8). MS of product B2 gave greatest intensity of EB

major isomer of the substitution product was obtained with each reagent tested. Those substitution compounds derived from the l3-hydroperoxide were identified as: l3-methoxy-12oxo-cis-9-octadecenoic acid (B 1) from reaction with methanol, l3-ethylthio-12-oxo-cis-9-octade cenoic acid (B 2 ) from ethanethiol, 1301eoyloxy-12-oxo-cis-9-octadecenoic acid (B3) from oleic acid. Since minor amounts of the 9-hydroperoxide (21 %) were present in the substrate, the corresponding isomer of B 1, B2 and B 3 also was observed as detected by characteristic fragment ions in MS, although in some samples the corresponding fragment ions for this isomer were barely detectable. Elution volumes of B 1, B2 , and B 3 upon chromatography by the silicic acid column regime outlined LIPIDS, VOL. 10, NO.8

mle 145 ion (CH3-[CH2] 4-ethane thiol>methanol>water. Since reaction rates are concentration dependent and each of the nucleophiles tested was in considerable excess (approached CO at completion of the reaction), the ability of each reagent to substitute in preference to H2 0 can be emphasized by including the effect of concentration in the mole ratio calculation as follows: Bn/C~:A/C~~o, where Bn is moles of the substitution co~pound produced; C~ is the initial molar concentration of the reagent; A is moles of a:-ketol; and C~~o is the initial molar concentration of H~ 0 (Table III). As can be seen from these calculated values in Table III. the order of reactivity is accentuated. In othe~ experiments when oleic acid, ethanethiol and methanol each was reacted in equimolar concentrations (0.034 M), the mole ratio of B2 to A was even lower (0.30) for the ethanethiol

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reaction than given in Table III; no B1 product could be detected with the methanol reaction. In this manner, the relative reactivity of the three reagents was confirmed. All the isomerase reactions occurred largely without problems, except those catalyzed in the presence of ethanethioi. Use of ethanethiol leads to significant (presumably nonenzymatic) reduction of linoleic acid hydroperoxide to the corresponding hydroxyoctadecadienoic acid, but since this side reaction is parallel to the substitution reactions of interest and the reagent is present in considerable excess, the desired comparison of the reactivity of the various nucleophiles in the course of the isomerase reaction is not compromised seriously. ACKNOWLEDGMENTS Mass spectral analysis was done by R. Kleiman, and NMR analysis was done by D. Weisleder.

REFERENCES 1. Zimmerman.

2. 3. 4. 5. 6. 7. 8.

9. 10.

D.C.. Biochem. Biophys. Res. Commun. 23:398 (1966). Veldink. G.A., J.F.G. Vliegenthafl, and J. Boldingh, FEBS Lett. 7: 188 (1970). Gardner, H.W., J. Lipid Res. 11:311 (1970). Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall, 1. BioI. Chern. 193:265 (1951). Gardner, H.W., K. £Skins, G.W. Grams, and G.E. Inglett, Lipids 7: 324 (1972). Schlenk, H., and J.L. Gellerman, Anal. Chern. 32: 1412 (1960). Gardner, H.W., R. Kleiman, and D. Weisleder, Lipids 9:696 (1974). Silverstein, R.ivl., and G. Clayton Bassler, "Spectrophotometric Identification of Organic Compounds," John Wiley and Sons, New York, N.Y., 1963. Zimmerman, D.C., B.A. Vicl" and T.K. Borg, Plant Physiol. 53: 1 (1974). Graveland, A., Lipids 8: 599 (1973).

[Received February 6, 1975]

LIPIDS, VOL. 10, NO.8