THE JOUIWAL OF BIOLOGICAL CHEMIBTRY

Vol. 246, No. 16, Issue of August 25, pp. 502.55030, 1971
Printedin U.S.A.

Biohydrogenation of Unsaturated Fatty Acids

VI. SOURCE OF HYDROGEN AND STEREOSPECIFICITY OF REDUCTION*

(Received for publicat,ion, March 9, 1971)

1. S. ROSENFELI) ANU S. B. TOVE~.

From the Department oj Biochemistry, North Camha State Uwiuersity, Raleigh, North Carolina 2760?

SUMMARY prepare. This report deals with the hydrogenation reaction with
intact cells in which the source of hydrogen and stereospecificity
The biohydrogenation of either linoleic acid or cis-9, trans-
of the reduction of the double bond were investigated.
11 ,cis-13-octadecatrienoic acid (punicic acid) by Butyriuibrio
jbrisolvens results in the formation of trans-11-octadecenoic EXPERIMENTAL PROCEDURE
acid. Incubation of whole cells with tritiated formate, triti-
ated succinate, and glucose labeled with tritium in various Bacterial Culture

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positions failed to result in the labeling of the monoenoic acid B. fibrisolvens strain A-38 was grown and maintained as pre-
product. In contrast, experiments performed in DzO indi- viously described (2) except that the oxidation-reduction poten-
cated that deuterium was incorporated at the cis double tial dye, resazurin, was not included and the media was gassed
bond(s) reduced by the microorganism. This reduction, with an atmosphere of oxygen-free 95% COZ and 50/O H, for 2
which takes place stereospecifically, was found to occur by hours prior to inoculation. The cells were harvested by centri-
cis addition to the D side of cis-9, tram-1 1-octadecadienoic fugation in 250~ml capped polypropylene bottles in a Sorvall
acid, an intermediate in the biohydrogenation of linoleic acid. GSA rotor at 14,600O X g for 15 min.
The distribution of deuterium at the reduced carbon atoms Chlorella vulgaris was grown and maintained as described by
shows an isotope effect and leads to the speculation that re- Harris and James (4).
duction occurs by addition of a proton and hydride ion medi-
Substrates
ated by an unknown carrier.
Linoleic and a-eleostearic acids were obtained from the Hormel
Institute. The tritiated substrate cis-9, trans-ll-[Q, lo-3H]
octadecadienoic acid was prepared by reduction of octadec-Q-
yn, trans-ll-enoic acid with tritium gas and was the generous
gift of Dr. L. J. Morris, Unilever, Shambrook, Bedford, Eng-
The pathway of biohydrogen:rtion of linoleic acid by the land.
anaerobic rumen bacterium, Butyrivibrio Jibrisolvens, consists of Punicic acid (c&Q, trans-11 ,cis-13-octadccatrienoic acid) was
at least two reactions: (a) an initial isomerization to L-9, isolated from the seed oil of Punica granatum (pomegranate)
trans-ll-octadecadienoic acid and (b) the subsequent hydrogena- purchased in a local market. The outer covers of the pome-
tion of this compound to trans-ll-octadecenoic acid (1, 2). granates were removed and the fruit was allowed to soak in water
Partial purification of linoleic acid isomerase, the enzyme that for 1 to 2 days. The fruit was then squeezed by hand to remove
cntalyzes the isomerization, has been achieved and some of its the fleshy coating; and the small, hard, white seeds were dried
properties have been investigated. It shows marked specificity in a vacuum desiccator over PZOS. The seeds were ground
for a free carboxyl group and a cis-Q,cis-12 pentadiene system in a Wiley Mill and extracted under nitrogen with petro-
(3). These studies were greatly facilitated by the finding that leum ether (b.p. 40-60) in a Soxhlet apparatus for 24 hours.
this reaction takes place under aerobic conditions. In contrast
The acid was isolated by low temperature crystallization as de-
to this, t,he hydrogenation reaction appears to be obligately an- scribed by Crombie and Jacklin (5). The white crystalline
aerobic, and active cell-free preparations have been difficult to product melted at 43 (lit. m.p. 40-42) (5) and gave the ex-
* This work is a contribution from the Department of Bio- pected ultraviolet spectrum with maxima at 264, 274 and 285
chemistry, School of Agriculture and Life Sciences and School of nm.
Physical and Mathematical Sciences. It is Paper 3421 of the Jour- The alcohol derivative of punicic acid was prepared from the
nal Series of the, North Carolina State University Agricultural
Experiment Station, Raleigh, North Carolina. This work was methyl ester by treatment with LiAIHt (6). The alcohol gave
sunnorted in oart bv Public Health Service Research Grant AM- the same absorption spectrum as punicic acid and migrated as
02483 from &e Naiional Institute of Arthritis and Metabolic a single spot on thin layer chromatoplates of silica gel with
Disecxs. High resolution mass spectrometry was done at the heptane-isopropyl ether-acetic acid (6 :4 :0.3). The infrared ;spec-
Research Triangle Institute Cenier for Mass Spectrometry under trum exhibited characteristic peaks at 3600.0 cm-1 (OH) and
Grant PR 330 from the Biotechnology Resources Branch of the
National Institutes of Health. at 981.4 aud 932.0 cm- (cis, truns-conjugateddoublebond sys-
t To whom correspondence should be addressed. tern). No peaks were observed in t,he carbonyl region.

5025
5026 Source of Hydrogen and Stereospecificity of Reduction Vol. 246, No. 16

c1a320 Isolation of Reaction Products-Following incubation, the reac-

Nuclidic mass Calculated: 264.2453 tion mixture was extracted according to the method of Dole (9).
Found : 264.2448 The products of the fatty acid substrates were methylated by
diazomethane and the monoenoic acids were isolated by chroma-
The ci.s-9, trans-11 , cis-13-octadecatriene was prepared by tography of their methyl esters on silicic acid-silver nitrate
LiAIHd reduction of the mesylate ester of the alcohol (7, 8). The columns (10, 11). In each case, a single component was ob-
hydrocarbon had an absorption spectrum identical with that of served when examined by gas-liquid chromatography. When
punicic acid and gave a single spot when chromatographed on the alcohol or paraffin derivatives of punicic acid were used as
silica gel plates with hexane as the solvent. When subjected to substrates, their hydrogenation products were separated by
gas-liquid chromatography, a single peak was observed. The chromatography on Florisil (12). The monoene paraffin prod-
infrared spectrum showed no peaks in the carbonyl region, but uct was indicated by its retention time during gas-liquid chro-
the same doublet, characteristic of the cis-trans double bond sys- matography with 9-nonadecene and S-heptadecene as standards.
tem, was observed. The monoene alcohol was indicated by its cochromatography
with trans-11-octadecenol on silicic acid-silver nitrate thin layer
plates (13).
Nuclidic mass Calculated: 248.2504 Stereospecijicity Studies-In these studies cis-9, trans-ll-
Found : 248.2509 [9,10-3H]octsdecadienoic acid was used as the substrate. The
Deuterium oxide was supplied by Stohler Isotope Chemicals labeled trans-11-octadecenoic acid was isolated, methylated, and
and the acid hydrolysate of algae cells grown on D20 was obtained reduced to methyl stearate by hydrazine (14). After saponifica-
from Merck. tion, l-14C-stearic acid was added and the doubly labeled stearic
Tritiated sodium formate, 2, 3-3H-succinic acid, and 5-3H- acid incubated with a suspension of Chlorella as described by
glucose were obtained from Amersham-Searle. Glucose labeled Morris et al. (15). The algal suspension was then extracted

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with tritium in positions 1, 2, 3, and 6 was obtained from New with chloroform-methanol (2: 1)) and the methyl esters of the
England Nuclear. fatty acids were prepared by transmethylation (16). Methyl
The standard paraffins, 9-nonadecene and %heptadecene, were oleate and methyl linoleate were isolated by argentation column
obtained from the Chemical Samples Company. chromatography (11). Each gave a single peak upon gas-liquid
chromatography.
Methods To ensure that the tritium label had not moved during the hy-
drogenation of the Ag-bond, l-14C-labeled stearic acid was omitted
Incubations-A solution of 5 mg of the fatty acid or derivative from the Chlorella incubation and the tritiated oleic acid was
in benzene was added to a 125-ml Erlenmeyer flask and the isolated from the Chlorella suspension as previously described.
solvent was removed with a stream of nitrogen. After the Carrier methyl oleate was added and reductive ozonolysis was
benzene had evaporated, 12 ml of 0.05 M potassium phosphate accomplished by the method of Edwards (17), except that the
buffer, pH 6.6, containing 0.48 g of bovine serum albumin (Frac- 2,4-dinitrophenylhydrazine reagent of Johnson (18) was used.
tion V) was added. Twelve milliliters of a bacterial suspension The dinitrophenylhydrazone derivatives of the aldehyde and
in 0.1 M phosphate buffer, pH 6.6, were added and the flask was aldehydo-ester fragments were separated by chromatography on
stoppered with a rubber stopper equipped with two short glass alumina (19). The purity was established by the single spot
tubes, on which were placed 2-inch pieces of thin walled rubber obtained for each fragment when chromatographed on thin layer
tubing. The flasks were placed in an ice bath and flushed with plates of Microcel-T38 (20). To determine the tritium in each
hydrogen for 20 min, after which the rubber tubes were closed fragment, the nonanal-dinitrophenylhydrazone and the methyl-
with a pinch clamp. Incubation was carried out with gentle 9-oxononanoate dinitrophenylhydrazone were completely oxi-
agitation for 4 hours at 37. dized (21) and the tritiated water was absorbed in 20 ml of a
Undue exposure to air was avoided during the preparation of solution of 30% methanol in toluene that contained 6 g of Omni-
the bacterial suspension. Following centrifugation, the bacterial fluor (New England Nuclear) per liter.
pellet was suspended in 13 ml of 0.1 1\~phosphate buffer, pH 6.6, Oxidative cleavage of the 3H-labeled methyl oleate to nonanoic
that had been thoroughly flushed with hydrogen. The tube acid and monomethyl azelaic acid was accomplished according
containing the cells was flushed with hydrogen for 5 min, stop- to the procedure of Castle and Ackman (22). The nonanoic
pered, and shaken to disperse the bacteria. The suspension was acid was isolated by steam distillation and the monomethyl
diluted with thoroughly gassed buffer such that a 1: 100 dilution azelaic acid was isolated by thin layer chromatography on
gave an absorbance of 1 at 420 nm. silica gel plates withheptane-isopropyl ether-acetic acid (6:4 :0.3).
When the tritium-labeled substrates were used, 100 PCi were The monocarboxylic acid was extracted with ether and trans-
added as an aqueous solution to the buffered albumin. When ferred to a counting vial. The spot corresponding to the mono-
cis-9, truns-11[9, 10-3H]octadecadienoic acid was incubated, vol- methyl azelaic acid was scraped off and the product was eluted
umes one-third the usual size were used. with methanol and counted.
In experiments in which DzO was used, the buffer solution Mass Spectrometry-Following extraction, methylation, and
was evaporated to dryness and the buffer salts were dissolved in isolation of the product of either a deuterated substrate or fatty
the appropriate volume of DZO. acid substrate incubated in DzO, mass spectra were obtained by
In experiments conducted with the alcohol or paraffin deriva- means of a AEI-12 mass spectrometer.
tive of punicic acid, the substrate was dispersed by sonic oscil- The 11,12-dimethoxy methyl octadecanoate derivative of the
lation (Branson) in a small amount of buffer prior to incuba- methyl truns-11-octadecenoate obtained from the incubation of
tion. linoleic acid with B. jibrisolvens in DzO was prepared and isolated
Issue of August 25, 1971 I. X. Rosenfeld and S. B. Tove

as described by Neihaus and Ryhage (23). The monoene frac- TABLE I

tion from the incubation of punicic acid in D20 was reduced to Recovery of aH frqm products of &saturation of doubly labeled
the paraffin via the alcohol and mesylate ester (7,8), as previously stearic acid by Chlorella vulgaris
described and oxidatively cleaved by a modified method of Experiments with cis-9, trams-11[9, 10-3Hloctadecadienoic acid
Scheuerbrandt and Block (24). Since the paraffin was insoluble were as described in the text. The biohydrogenation product
in their reaction mixture, the solvent was removed and the was reduced to stearate and incubated with Chlorella. Oleic and
the following solutions were added per 5 mg of unsaturated hy- linoleic acids were isolated and counted.
drocarbon: 0.8 ml of t-butyl alcohol, 0.3 ml of a mixture of 0.02 Experiment and acid =H C SH: C
M Khln04 and 0.19 M NaI04, 0.12 ml of 0.04 M K&Ox, and
apm x 10-z dJ%Pz x NJ-
finally 0.6 ml of water. The flask was sealed and stirred for 2
hours at room temperature and the acid fragments were isolated 1. Substrate 18:O~. . 501.8 149.3 3.35
(24). Mass spectra of their methyl esters were obtained by Product 18: 1. . 35.3 11.2 3.15
Product 18:2.. _. . 6.6 2.0 3.30
using the gas chromatographic inlet system of a model 9000
2. Substrate 18:O.. . 467.5 40.6 11.50
LKB mass spectrometer. A four-foot column of ethylene glycol
Product 18: 1. 243.1 23.3 10.40
succinate-HaPOd was used with temperature programming. Product 18:2. . . 28.0 2.5 11.20
Several scans were obtained for all samples and the peaks of
interest were corrected for natural abundance. a The number to the left of the colon represents the number of
Gus-Liquid Chromatography--The methyl esters of the acids carbon atoms in the chain; the number to the right of the colon
obtained from incubations with linoleic acid, cr-eleostearic acid, designates the number of double bonds.
and the c&runs-conjugated acid mixture were analyzed by
gas-liquid chromatography. The paraffins isolated from incu- presence of the trans bond at C-13 prevented the hydrogenation
bation of B. jibrisolvens with cis-9, truns-11 , cis-13-octadecatriene of the cis-9 bond.

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were also subjected to gas-liquid chromatography. An F and M Hydrogenation of Parafin and Alcohol Derivatives of Punicie
model 700 flame ionization instrument equipped with four-foot Acid--Gas-liquid chromatography of the hydrocarbons isolated
columns of 10% diethylene glycol succinate on Chromosorb W after incubation of B. Jibrisolvens with cis-9, truns-11 ,cis-13-
was used. octadecatriene showed the appearance of a peak not observed in
Other Analytical Procedures-Ester groups were determined by the hydrocarbon fraction from a zero time control. This peak,
the procedure of Snyder and Stephens (25). amounting to 21.5% of the hydrocarbon fraction, exhibited a
Radioactivity was measured in a Packard Tri-Carb liquid retention time corresponding to that calculated for an octa-
scintillation spectrometer by usin, 0 a scintillation solution of decene.
Omnifluor (New England Nuclear) in toluene (4 g per liter). The alcohol derivative of punicic acid also appears to be re-
Infrared spectra were measured in a Beckman IR-8 in carbon duced, since analysis of the reaction products by argentation
disulfide solution. thin layer chromatography showed a spot that corresponded to
Jloleculnr formulas were determined by accurate mass meas- truns-11-octadecenol.
urement on a MS-902 mass spectrometer. Stereospecijcity of Biohydrogenation Reaction-Stereospecific
desaturation of stearic acid by C. vulgaris (15) provided the
RESULTS
rationale by which the stereospecificity of the reduction of the
Hydrogenation of Punicic Acid---Linoleic acid isomerase, the cis-9 double bond of cis-9, truns-ll-octadecadienoic acid was
enzyme that catalyzes the first reaction in the biohydrogenation studied. In these experiments cis - 9, truns - 11[9,10 - 3H]octa-
pathway, has marked substrate specificity requirements (3). decadienoic acid was incubated with B. $brisolvens. The la-
Since B. fibrisolvens was able to hydrogenate a mixture of cis- beled truns-11-octadecenoate product was isolated as the methyl
frans conjugated dienes (A9~11,A1J,1z,A8~10)(l), it appeared that the ester and converted to stearic acid. Following the addition of
specificity properties for the hydrogenation reaction were likely lJ4C-stearic acid, the doubly labeled stearic acid was incubated
to be less stringent. Accordingly, the naturally occurring con- with Chlorella. In each of two experiments, the ratio of 3H:14C
jugated octadecatrienoic acid, punicic acid, with a cis-9, truns- in the oleic and linoleic acids isolated from the algae was the
11 ,&s-13 double bond system seemed likely to serve as a sub- same as the 3H:14C of the stearic acid substrate (Table I). To
strate. When punicic acid was incubated with the bacteria, ensure that migration of the labeled hydrogens had not occurred
analysis of the methyl esters of the free fatty acids isolated from during incubation with B. Jibrisolvens, stearic acid containing
the incubation mixture showed a complete disappearance of the only the tritium label was incubated with Chlorella. The dini-
conjugated triene and the appearance of a peak coincident with trophenylhydrazone derivatives of the aldehyde fragments ob-
methyl oleate. After isolation of this product by argentation tained from reductive ozonolysis of the tritiated methyl oleate
chromatography, it was subjected to analysis by infrared spec- were found to contain almost equal amounts of tritium (Table
troscopy and mass spectrometry. In each case the spectra ob- II). Another portion of the labeled oleate was oxidatively
tained were identical with those of the trans-ll-octadecenoate cleaved. No radioactivity was observed in either the nonanoic
product of the linoleic acid incubation. Moreover, reductive or monomethyl azelaic acid fragments. These results show that
cleavage of the methyl ester yielded heptaldehyde and methyl- the tritium in the cis-9, truns-1119, 10-3H]octadecadienoate had
1 l-osoundecanoate, which indicated the position of unsatu- remained at positions 9,lO during incubation with B. fibrisol-
ration to be at C-11. vens.
In contrast to punicic acid, cis-9, trans-11 , trans-13-octadec- Source of Hydrogen in Hydrogenation Reaction---Initial at-
atrienoic acid (a-eleostearic acid) was not changed during incu- tempts to ascertain the source of the reducing hydrogen were
bation with the bacteria. Thus, it would appear that the made by incubating B. jibrisolvens with a series of tritiated
5028 Source of Hydrogen and Stereospecificity of Reduction Vol. 246, No. 16

TABLE II TABLE V
Trilium. in reductive ozonolysis fragments of methyl oleate isolated Deuterium cgntent of fragments of methyl end and carboxyl end of
S:orn Zhlorella after incubation with cis-9, trans-11[9,1 O-aH]- 11 ,I$-dimethoxu octadecanoate prepared from methyl trans-il-
octaclecadienoic acid with Butyrivibrio jibrisolvens octadecenoate isolated after linoleic acid incubation with
The oeonide of methyl oleate was reduced with 2,4-dinitro- Butyrivibrio $brisolvens
phenylhydrazine and the dinitrophenylhydrazones of nonanal Mass spectra (70 e.v.) were obtained with an AEI-12 spectrome-
and methyl 9-oxononanoate were oxidized and the water from ter and the peaks of interest were corrected for natural abundance.
each was collected and counted. The specific activity of the 9, lo-
di-3H-cis-9, trans-11-octadecadienoic acid was 60 mCi per mmole. Per cent of parent ions containing

Fragment Tritium No D atoms / 1 D atom 1 2Datoms

&5m/Jmw1e x 10-a
Nonanal................................. 100.0
Methyl 9-oxononanoate. . . 80.0

TABLE VI
TABLE III Distribution of deuterium in trans-li-octadecene prepared from
Incorporation of 3H from 1 -3H-glucose and VH-glucose into trans- methyl trans-11 -octadecenoate product of linoleic and punicic
11 -octadecenoic acid and the saturated fatty acids by acid incubations
Butyrivibrio jibrisolvens After isolation, methyl trans-11-octadecenoate was converted
Incubations were carried out with linoleic acid and the labeled to the paraffin derivative and oxidatively cleaved to heptanoic
glucose as described in the text. The methyl esters of the satu- and undecanoic acids. The methyl esters of these acids were
rated fatty acids and trans-11-octadecenoic acid were isolated by subjected to gas-liquid chromatography-mass spectrometer

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silicic acid-silver nitrate column chromatography. The frac- analyses on a LKB model 9000 spectrometer (70 e.v.). The peaks
tions emerging from the column first were taken as saturated fatty of interest were corrected for natural abundance. The positions
esters. Ester concentrations were determined on a portion of refer to the original trans-11-octadecenoic acid, positions 9 and 10
the sample, and another portion was counted in a liquid scintilla- coming from undecanoic acid and positions 13 and 14 coming from
tion spectrometer. Radioactivity measurements were corrected heptanoic acid.
for background and quench. The specific activity of each trit-
iated glucose was 100 $Zi per mmole. Deuterium at positions
Substrate
Substrate Saturated acids trans-11.18: 1 1 9 1 10 1 13 / 14

cpm//mde C@&/j.Hde

PH-Glucose 7279 40 cis-cis-18:2 (A9.12)

3-3H-Glucose. 366 58 cis-trans-&s-18:3 (As.ll.lr)

TABLE IV ments in which approximately 3 ml of bacterial pellet were

Deuterium in methyl trans-11 -octadecenoate isolated after incubation suspended in 12 ml of 99% DzO indicate that, during reduction
of linoleic.acid and punicic acid of the cis-9, trans-11-octadecadienoic acid in DzO, 2 additional
atoms of deuterium were present in the resulting truns-ll-
Per cent of parent ions containing
Ex- octadecenoic acid (Table IV). The actual level of deuterium
Substrate peri-
ment XoD 1D 2D 3D 4D incorporated reflects not only the specific activity of the water
atoms atom atoms atoms atoms but the rate of equilibration of deuterium with the active hydro-
~-__-
gens of the bacterial cell.
cis, &s-18:2 (Agn12) 1 15 33 43 9 0 Similar experiments were performed with punicic acid as a
2 15 16 60 7 0
substrate. Since punicic acid does not undergo isomerization
3 9 40 39 3 0
cis, trans,cis-18:3 (Ag.11v13) 1 7 19 34 27 13 prior to its hydrogenation to the truns-11-monoene and since
2 8 24 36 25 7 both cis bonds are reduced, it was expected that 4 deuterium
atoms would be incorporated. The results in Table IV show
this to be the case.
substrates. No tritium was incorporated from glucose labeled These experiments analyzed by mass spectrometry indicated
in positions 1>2 73 95 9 and 6 or from tritiated succinate or formate. that deuterium was incorporated during the reduction of the
,Evidence that l-3H-glucose and 3-3H-glucose were metabolized cis double bond(s) but did not reveal the positions of substitu-
as expected, i.e. provided reducing equivalents for fatty acid tion. To localize the incorporated deuterium, the methyl ester
synthesis, comes from the observation that tritium was found in of truns-11-monoenoic acid resulting from linoleic incubation
the fatty acids synthesized by the cell (Table III). was converted to the 11,12-dimethoxy derivative and subjected
To determine whether or not water provides the hydrogens to mass spectrometry (23). When treated in this manner, the
for reduction, incubations of I?. jlbrisolvens in DzO were per- dimethoxy compound undergoes cleavage between the meth-
formed. When incubated in D20, a single deuterium atom was oxyl groups, yielding 2 ions (m/e 129 and m/e 229) corresponding
found to be incorporated at C-13 during the isomerization of to the methyl end and the carboxyl end of the methyl truns-ll-
linoleic acid to cis-9, truns-11-octadecadienoic acid (2), but the octadecenoate (23). The results (Table V) indicate that the deu-
hydrogenated product was not examined. More recent experi- terium atoms incorporated during hydrogenation of the A-9,
Issue of August 25, 1971 I. X. Rosenfeld and S. B. Tove 5029

trans-11-octadecadienoic acid were located in the carboxyl por- acid were hydrogenated and, thus, support this conjecture.
tion of the molecule. Some naturally occurring compounds that contain a truns-con-
The distinct positions of substitution were obtained by reduc- jugated double bond system, such as the carotenes, escape
ing the deutcrated truns-11-octadecenoic acid from the punicic hydrogenation in the rumen (27). The findings with cu-eleo-
acid and linoltic acid incubations to the trans-11-octadecene. stearic acid and the punicic acid derivatives suggest that it is
Oxidative cleavage and mass spectrometry of the methyl esters the presence of the truns configuration rather than the absence
of the heptanoic acid and undecanoic acid fragments allowed of a carboxyl group which accounts for their lack of hydrogena-
the use of the McLafferty rearrangement to determine the tion.
location of deuterium in the original truns-1 l-octadecenoic acid. Experiments with tritiated glucose (labeled in positions 1, 2, 3,
The major peak of methyl esters longer than Cs is due to a 5, and 6) showed that the hydrogen that reduces the double bond
rearranged ion of m/e 74. This ion contains 3 hydrogen atoms: did not come directly from glucose. Similarly, absence of tritium
2 from the a-carbon and 1 from the y-carbon of the fatty acid incorporation in truns-11-octadecenoic acid from labeled formate
methyl ester (26). In this case, the two hydrogens bonded to and succinate, as well as the absence of deuterium incorporation
the a-carbon of the methyl heptanoate fragment represent the from a totally deuterated algal hydrolysate, indicated that the
hydrogen atoms at C-13 of the truns-11-octadecenoic acid. direct addition of hydrogen from an organic substrate was un-
Those bonded at C-10 of the trans-11-octadecenoic acid would likely. In contrast, the fact that 2 deuterium atoms were incor-
correspond to a-hydrogens of the methyl undecanoate fragment. porated from D,O during the hydrogenation of cis-9, truns-ll-
The appearance of a large peak at m/e 75 in the spectrum of octadecadienoic acid and 4 deuterium atoms from DzO were
each of the monocarboxylic methyl esters from both substrates incorporated in the biohydrogenation of punicic acid indicates
indicates that hydrogen from HZ0 is incorporated at C-10 of that water is the immediate source of hydrogens used to reduce
linoleic acid and at C-10 and C-13 of punicic acid. the cis bond(s). These results, however, do not preclude the
From the ratio of the m/e 74 ion to the m/e 75 ion and the direct reduction of a carrier by an organic substrate if the hydro-

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assumption that all of the deuterium incorporated was bonded gen carrier can undergo rapid exchange with water.
to the carbons of the cis double bond(s), the distribution of Examination of the isotope distribution in the reduced products
deuterium at each of the positions of the double bond could be showed that the position(s) adjacent to the truns double bond
calculated. The results (Table VI) show that the carbons adja- contains less deuterium than the distal position(s). This dis-
cent to the truns double bond contain less deuterium than those tribution indicates that discrimination against deuterium had oc-
distal to the truns bond. From the mass peaks associated with curred at C-10 of cis-9,truns-ll-octadecadienoic acid, and at (-10
the parent ions, it may be calculated that 3056 of the hy-drogen and C-13 of punicic acid. Therefore, the hydrogens added at
atoms at C-13 and C-14 and 28T1 of the hydrogen at C-9 and C-10 or C-13 must have experienced at least one more bond-
C-10 were replaced by deuterium. These results, together with breaking event than those added at C-9 or C-14. These results
the similarity in distribution, suggest that both of the cis bonds lead to the suggestion that the mechanism of biohydrogenation
of punicic acid were hydrogenated by the same system. involves the addition of a proton to the cis bond at the position
distal to the truns bond and that reduction of the double bond is
DISCUSSION
finally completed by a hydride ion provided by an unknown car-
The hydrogenation of linolcic acid initially involves the isom- rier.
erization of linoleic acid to a cis-9, truns-1 l-octadecadienoic Since ferredoxin occurs commonly in anaerobic organisms, one
acid. Several reports on the natire and characteristics of lino- might expect this electron carrier to be involved in biohydrogena-
leic acid isomerase, the enzyme that catalyzes this reaction, tion. However, we were unable to observe a ferredoxin band on
have been published (2, 3), but, until now, none of the findings a DEAE-cellulose column following chromatography (28) of cell
concerning the reduction of the conjugated intermediate to truns- extracts of B. $brisolvens.
11.octadecenoic acid have been reported. Upon biohydrogenation and reduction of the monoenoic acid to
As there is no readily available source of this cis-9,truns-ll- stearic acid, it is possible, with the stearic acid as a substrate for
octadecadienoic acid intermediate, punicic acid, cis-9, truns-ll , Chlorellu, to determine the stereochemistry of hydrogen addition
cis-13.octadecatrienoic acid represents a unique substrate which by B. jibrisolvens. Morris has used this approach to study the
facilitates the investigation of t,he reductive reaction. It has stereospecificity of the biohydrogenation of oleic and elaidic acids
been shown (Table IV) that both cis double bonds are hydro- by mixed rumen flora (29), and Schroepfer, using Corynebucterium
genated, resulting in the same product as that obtained from diphtheriue instead of ChZoreZZu, determined the stereospecificity
linoleic hydrogenation. It is interesting to note that, when of the hydroxylation of oleic acid (30). As reported in the pre-
ar-eleostearic acid (cis-9, truns-11 , trans.13.octadecatrienoic acid) vious paper of this series, the same approach was used to show
is used as a substrate, no reaction occurs. The inactive a- the stereospecificity of hydrogen addition at C-13 of linoleic acid
eleostearic acid is a conjugated triene similar to punicic acid during its isomerization (31).
differing only in that the configuration of the Al3 bond is truns If the biohydrogenation of cis-9, truns-11-octadecadienoic acid
instead of cis. It is apparent, therefore, that the configuration occurs by cis addition, then either DD or LL-9, 10-di-aH-truns-ll-
of the conjugated truns double bond system imparts a degree of octadecenoic acid would result. Desaturation by Chore&x of the
alteration to the molecule such that the organism is incapable of stearic acid derived from the truns-ll-octadecenoic acid would
reducing the cis bond of the conjugated triene. yield oleic and linoleic acids showing either complete recovery of
The similarity of deuterium distribution at both cis bonds the tritium for the D-labeled enantiomer or complete loss of trit-
indicates that, unlike linoleic acid isomerase, the carboxyl group ium for the L-labeled enantiomer. The truns addition of hydro-
is a dispensable feature of the substrate. Preliminary experi- gen by B. fibrisolvens would yield threo-di-aH-truns-ll-octudccc-
ments showed that the alcohol and paraffin derivatives of punicic noic acid, and the oleic acid isolated from Chlorellu would be
5030 Source of Hydrogen and SkreospeciJicity of Reduction Vol. 246, No. 16

expected to show one-half of the tritium label. The results (Ta- 7. BAUMANN, W. J., AND MANGOLD, H. K., J. Org. Chem., 29,
ble I) showed complete recovery, and reductive and oxidative 3055 (1964).
8. BAUMANN, W. J., JONES. L. L.. BARNUM. B. E.. AND MANGOLD.
ozonolysis of the oleic acid showed that the tritium label had not H. K., &em. khys. Lipids, i, 63 (1966).
moved during incubation. We conclude, therefore, that the bio- 9. DOLE, V. P., J. Clin. Invest., 36, 150 (1956).
hydrogenation of L-9, trns-1 I-octadecadienoic acid by B. 10. SCHLENIC, H.. AND GELLERMAN. J. L.. Anal. Chem.. 32. 1412
fibrisolvens occurs by cis addition to the D side of carbons 9 and 10. (1960):
11. DEVRIES, B., J. Amer. Oil Chem. Sot., 40, 184 (1963).
Morris (29) has shown that the biohydrogenation of oleic acid 12. CARROLL, K. K., J. LimZ Res.. 2. 135 (1961).
involves cis addition to the L side. However, B. fibrisolvens is 13. MORRIS, L. J., in A. I!. JAM& AAD L.J. I~~ORRIS (Editors),
unable to hydrogenate oleic acid (32). Consequently, although New biochemical separations, D. Van Nostrand, New York,
the biohydrogenation of oleic and &s-9, trans-11-octadecadienoic 1964, p. 300.
14. ROEHM, J. N., AND PRIVETT, 0. S., J. Lipid Res., 10,245 (1969).
acids is similar in that both involve cis addition to a cis double 15. MORRIS, L. J., HARRIS, R. V., KELLY, W., AND JAMES, A. T.,
bond, it is clear that, the two systems are different. Biochem. J., 109, 673 (1968).
Studies with a cell-free system capable of carrying out biohy- 16. TOVE, S. B., J. Nutr., 76, 361 (1961).
drogenation are in progress. Particular effort is being directed 17. EIIWARDS, H. M., JR., Lipids, 1, 1 (1966).
toward the elucidation of the nature of the electron donor and 18. JOHNSON, G. D., J. Amer. Chem. Sot., 73, 5888 (1951).
carrier.
19. SCHWARTZ, D. P., WEIHRAUCH, J. L., AND BURGWALD, L. H.,
Anal. Chum., 4i, 984 (1969).
20. SCHWARTZ, D. P.. SHAMEY. J.. BREWINGTON. C. R.. AND PARI~S.
Acknowledgments-We wish to thank Dr. Marion Miles of the 0. W., ikicrochem. J., i3, 407 (1968).
21. PETERSON, J. I., Anal. Biochem., 31, 189 (1969).
Department of Chemistry for some of the mass spectrometric 22. CASTLE, J. D., AND ACKMAN, R. G., Can. J. Chem., 45, 1405
analyses. We also thank Drs. D. P. Schwartz and 0. W. Parks (1967).
of the USDA, Washington, D. C., for helping us separate the 23. NEIHAUS, W. J., JR., AND RYHAGE, R., Anal. Chem., 40, 1840
2,4-dinitrophenylhydrazone derivatives and further appreciation (1968).

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24. SCHEUERBRANDT, G., AND BLOCK. , K.. , J. Biol. Chem., 237,
is extended to Dr. Parks for his help in gas-liquid mass spec- 2064 (1962).
trometry. We are also indebted to Dr. L. J. Morris for his many 25. SNYDER, F., AND STEPHENS, N., Biochim. Biophys. Acta, 34,
helpful comments and discussions. 244 (1959).
26. RYHAGE, R., AND STENHAGEN, E., in F. W. MCLAFFERTY
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