THE JOUIWAL OF BIOLOGICAL CHEMIBTRY

Vol. 246, No.

16, Issue

of August

Printedin

25, pp.

502.55030,

Biohydrogenation
VI. SOURCE

1971

U.S.A.

of Unsaturated

OF HYDROGEN

1. S. ROSENFELI)

ANU

From the Department

AND

S. B.

Fatty

STEREOSPECIFICITY

Acids

OF REDUCTION*
(Received for publicat,ion,

March

Raleigh,

2760?

9, 1971)

TOVE~.

oj Biochemistry,

North

Camha

Carolina

EXPERIMENTAL

Bacterial

PROCEDURE

Culture

B. fibrisolvens strain A-38 was grown and maintained as previously described (2) except that the oxidation-reduction
potential dye, resazurin, was not included and the media was gassed
with an atmosphere of oxygen-free 95% COZ and 50/O H, for 2
hours prior to inoculation.
The cells were harvested by centrifugation in 250~ml capped polypropylene
bottles in a Sorvall
GSA rotor at 14,600O X g for 15 min.
Chlorella vulgaris was grown and maintained
as described by
Harris and James (4).
Substrates
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-Qyn, trans-ll-enoic
acid with tritium gas and was the generous
gift of Dr. L. J. Morris, Unilever, Shambrook,
Bedford, England.
Punicic acid (c&Q, trans-11 ,cis-13-octadccatrienoic
acid) was
isolated from the seed oil of Punica granatum (pomegranate)
purchased in a local market.
The outer covers of the pomegranates were removed and the fruit was allowed to soak in water
for 1 to 2 days. The fruit was then squeezed by hand to remove
the fleshy coating; and the small, hard, white seeds were dried
in a vacuum desiccator over PZOS. The seeds were ground
in a Wiley Mill and extracted under nitrogen
with petroleum ether (b.p. 40-60) in a Soxhlet apparatus for 24 hours.
The acid was isolated by low temperature
crystallization
as described by Crombie and Jacklin (5). The white crystalline
product melted at 43 (lit. m.p. 40-42) (5) and gave the expected ultraviolet
spectrum with maxima at 264, 274 and 285
nm.
The alcohol derivative of punicic acid was prepared from the
methyl ester by treatment with LiAIHt (6). The alcohol gave
the same absorption spectrum as punicic acid and migrated as
a single spot on thin layer chromatoplates
of silica gel with
heptane-isopropyl
ether-acetic acid (6 :4 :0.3). The infrared ;spectrum exhibited characteristic peaks at 3600.0 cm-1 (OH) and
at 981.4 aud 932.0 cm- (cis, truns-conjugateddoublebond systern). No peaks were observed in t,he carbonyl region.

5025

Downloaded from www.jbc.org by guest, on October 8, 2012

The biohydrogenation
of either linoleic acid or cis-9, trans11 ,cis-13-octadecatrienoic
acid (punicic acid) by Butyriuibrio
jbrisolvens
results in the formation
of trans-11-octadecenoic
acid. Incubation
of whole cells with tritiated formate, tritiated succinate,
and glucose labeled with tritium in various
positions failed to result in the labeling of the monoenoic acid
product.
In contrast, experiments
performed
in DzO indicated that deuterium
was incorporated
at the cis double
bond(s)
reduced
by the microorganism.
This reduction,
which takes place stereospecifically,
was found to occur by
cis addition to the D side of cis-9, tram-1 1-octadecadienoic
acid, an intermediate
in the biohydrogenation
of linoleic acid.
The distribution
of deuterium
at the reduced carbon atoms
shows an isotope effect and leads to the speculation that reduction occurs by addition of a proton and hydride ion mediated by an unknown
carrier.

* This work is a contribution

from the Department
of Biochemistry, School of Agriculture
and Life Sciences and School of
Physical and Mathematical
Sciences. It is Paper 3421 of the Journal Series of the, North Carolina State University Agricultural
Experiment
Station, Raleigh, North Carolina.
This work was
sunnorted in oart bv Public Health Service Research Grant AM02483 from &e Naiional
Institute
of Arthritis
and Metabolic
Disecxs.
High resolution mass spectrometry
was done at the
Research Triangle Institute Cenier for Mass Spectrometry under
Grant PR 330 from the Biotechnology
Resources Branch of the
National Institutes of Health.
t To whom correspondence should be addressed.

North

prepare.
This report deals with the hydrogenation
reaction with
intact cells in which the source of hydrogen and stereospecificity
of the reduction of the double bond were investigated.

SUMMARY

The pathway of biohydrogen:rtion

of linoleic acid by the
anaerobic rumen bacterium, Butyrivibrio Jibrisolvens, consists of
at least two reactions: (a) an initial isomerization
to L-9,
trans-ll-octadecadienoic
acid and (b) the subsequent hydrogenation of this compound
to trans-ll-octadecenoic
acid (1, 2).
Partial purification
of linoleic acid isomerase, the enzyme that
cntalyzes the isomerization,
has been achieved and some of its
properties have been investigated.
It shows marked specificity
for a free carboxyl group and a cis-Q,cis-12 pentadiene system
(3). These studies were greatly facilitated by the finding that
In contrast
this reaction takes place under aerobic conditions.
to this, t,he hydrogenation
reaction appears to be obligately anaerobic, and active cell-free preparations
have been difficult to

State Uwiuersity,

5026

Source

Nuclidic

c1a320
Calculated:
mass
Found :

of Hydrogen

and Stereospecificity

264.2453
264.2448

The ci.s-9, trans-11 , cis-13-octadecatriene

was prepared
by
LiAIHd reduction of the mesylate ester of the alcohol (7, 8). The
hydrocarbon had an absorption spectrum identical with that of
punicic acid and gave a single spot when chromatographed
on
silica gel plates with hexane as the solvent.
When subjected to
gas-liquid chromatography,
a single peak was observed.
The
infrared spectrum showed no peaks in the carbonyl region, but
the same doublet, characteristic of the cis-trans double bond system, was observed.

Nuclidic

mass

Calculated:
Found :

248.2504
248.2509

Methods
Incubations-A
solution of 5 mg of the fatty acid or derivative
in benzene was added to a 125-ml Erlenmeyer
flask and the
solvent was removed with a stream of nitrogen.
After the
benzene had evaporated, 12 ml of 0.05 M potassium phosphate
buffer, pH 6.6, containing 0.48 g of bovine serum albumin (Fraction V) was added.
Twelve milliliters of a bacterial suspension
in 0.1 M phosphate buffer, pH 6.6, were added and the flask was
stoppered with a rubber stopper equipped with two short glass
tubes, on which were placed 2-inch pieces of thin walled rubber
tubing.
The flasks were placed in an ice bath and flushed with
hydrogen for 20 min, after which the rubber tubes were closed
with a pinch clamp. Incubation
was carried out with gentle
agitation for 4 hours at 37.
Undue exposure to air was avoided during the preparation
of
Following centrifugation,
the bacterial
the bacterial suspension.
pellet was suspended in 13 ml of 0.1 1\~phosphate buffer, pH 6.6,
that had been thoroughly
flushed with hydrogen.
The tube
containing the cells was flushed with hydrogen for 5 min, stoppered, and shaken to disperse the bacteria.
The suspension was
diluted with thoroughly gassed buffer such that a 1: 100 dilution
gave an absorbance of 1 at 420 nm.
When the tritium-labeled
substrates were used, 100 PCi were
added as an aqueous solution to the buffered albumin.
When
cis-9, truns-11[9, 10-3H]octadecadienoic
acid was incubated, volumes one-third the usual size were used.
In experiments in which DzO was used, the buffer solution
was evaporated to dryness and the buffer salts were dissolved in
the appropriate
volume of DZO.
In experiments conducted with the alcohol or paraffin derivative of punicic acid, the substrate was dispersed by sonic oscillation (Branson) in a small amount of buffer prior to incubation.

Vol.

246, No. 16

Isolation of Reaction Products-Following

incubation, the reaction mixture was extracted according to the method of Dole (9).
The products of the fatty acid substrates were methylated by
diazomethane
and the monoenoic acids were isolated by chromatography of their methyl esters on silicic acid-silver nitrate
columns (10, 11). In each case, a single component was observed when examined by gas-liquid
chromatography.
When
the alcohol or paraffin derivatives of punicic acid were used as
substrates,
their hydrogenation
products were separated by
chromatography
on Florisil (12). The monoene paraffin product was indicated by its retention time during gas-liquid chromatography with 9-nonadecene and S-heptadecene as standards.
The monoene alcohol was indicated by its cochromatography
with trans-11-octadecenol
on silicic acid-silver nitrate thin layer
plates (13).
Stereospecijicity
Studies-In
these studies cis-9, trans-ll[9,10-3H]octsdecadienoic
acid was used as the substrate.
The
labeled trans-11-octadecenoic
acid was isolated, methylated, and
reduced to methyl stearate by hydrazine (14). After saponification, l-14C-stearic acid was added and the doubly labeled stearic
acid incubated with a suspension of Chlorella as described by
Morris et al. (15). The algal suspension was then extracted
with chloroform-methanol
(2: 1)) and the methyl esters of the
fatty acids were prepared by transmethylation
(16). Methyl
oleate and methyl linoleate were isolated by argentation
column
chromatography
(11). Each gave a single peak upon gas-liquid
chromatography.
To ensure that the tritium label had not moved during the hydrogenation of the Ag-bond, l-14C-labeled stearic acid was omitted
from the Chlorella incubation
and the tritiated
oleic acid was
isolated from the Chlorella suspension as previously described.
Carrier methyl oleate was added and reductive ozonolysis was
accomplished by the method of Edwards (17), except that the
2,4-dinitrophenylhydrazine
reagent of Johnson (18) was used.
The dinitrophenylhydrazone
derivatives of the aldehyde and
aldehydo-ester fragments were separated by chromatography
on
alumina (19). The purity was established by the single spot
obtained for each fragment when chromatographed
on thin layer
plates of Microcel-T38
(20). To determine the tritium in each
fragment, the nonanal-dinitrophenylhydrazone
and the methyl9-oxononanoate
dinitrophenylhydrazone
were completely
oxidized (21) and the tritiated water was absorbed in 20 ml of a
solution of 30% methanol in toluene that contained 6 g of Omnifluor (New England Nuclear) per liter.
Oxidative cleavage of the 3H-labeled methyl oleate to nonanoic
acid and monomethyl
azelaic acid was accomplished according
to the procedure of Castle and Ackman (22). The nonanoic
acid was isolated by steam distillation
and the monomethyl
azelaic acid was isolated by thin layer chromatography
on
silica gel plates withheptane-isopropyl
ether-acetic acid (6:4 :0.3).
The monocarboxylic
acid was extracted with ether and transferred to a counting vial. The spot corresponding to the monomethyl azelaic acid was scraped off and the product was eluted
with methanol and counted.
Mass Spectrometry-Following
extraction,
methylation,
and
isolation of the product of either a deuterated substrate or fatty
acid substrate incubated in DzO, mass spectra were obtained by
means of a AEI-12 mass spectrometer.
The 11,12-dimethoxy
methyl octadecanoate derivative of the
methyl truns-11-octadecenoate
obtained from the incubation of
in DzO was prepared and isolated
linoleic acid with B. jibrisolvens

Downloaded from www.jbc.org by guest, on October 8, 2012

Deuterium
oxide was supplied by Stohler Isotope Chemicals
and the acid hydrolysate of algae cells grown on D20 was obtained
from Merck.
Tritiated
sodium formate, 2, 3-3H-succinic
acid, and 5-3Hglucose were obtained from Amersham-Searle.
Glucose labeled
with tritium in positions 1, 2, 3, and 6 was obtained from New
England Nuclear.
The standard paraffins, 9-nonadecene and %heptadecene, were
obtained from the Chemical Samples Company.

of Reduction

Issue

of August

I. X. Rosenfeld

25, 1971

RESULTS

Hydrogenation
of Punicic Acid---Linoleic
acid isomerase, the
enzyme that catalyzes the first reaction in the biohydrogenation
pathway, has marked substrate specificity requirements
(3).
Since B. fibrisolvens was able to hydrogenate
a mixture of cisfrans conjugated dienes (A9~11,A1J,1z,A8~10)(l), it appeared that the
specificity properties for the hydrogenation
reaction were likely
Accordingly,
the naturally occurring conto be less stringent.
jugated octadecatrienoic
acid, punicic acid, with a cis-9, truns11 ,&s-13 double bond system seemed likely to serve as a substrate.
When punicic acid was incubated with the bacteria,
analysis of the methyl esters of the free fatty acids isolated from
the incubation mixture showed a complete disappearance of the
conjugated triene and the appearance of a peak coincident with
methyl oleate. After isolation of this product by argentation
chromatography,
it was subjected to analysis by infrared spectroscopy and mass spectrometry.
In each case the spectra obtained were identical with those of the trans-ll-octadecenoate
product of the linoleic acid incubation.
Moreover,
reductive
cleavage of the methyl ester yielded heptaldehyde
and methyl1 l-osoundecanoate,
which indicated
the position of unsaturation to be at C-11.
In contrast to punicic acid, cis-9, trans-11 , trans-13-octadecatrienoic acid (a-eleostearic acid) was not changed during incubation with the bacteria.
Thus, it would appear that the

TABLE

Recovery of aH frqm products of &saturation

of doubly labeled
stearic acid by Chlorella vulgaris
Experiments
with cis-9, trams-11[9, 10-3Hloctadecadienoic
acid
were as described
in the text.
The biohydrogenation
was reduced
to stearate
and incubated
with Chlorella.
linoleic
acids were isolated
and counted.
Experiment

and acid

=H
apm

1. Substrate
Product
Product
2. Substrate
Product
Product
a The

18:O~.
18: 1.
18:2..
18:O..
18: 1.
18:2.

number

.
.
_. .
.
.

to the left

x 10-z

501.8
35.3
6.6
467.5
243.1
28.0

of the colon

product
Oleic and

SH: C

dJ%Pz x NJ-

149.3
11.2
2.0
40.6
23.3
2.5
represents

3.35
3.15
3.30
11.50
10.40
11.20
the number

of

carbon atoms in the chain; the number to the right of the colon
designates

the

number

of double

bonds.

presence of the trans bond at C-13 prevented the hydrogenation

of the cis-9 bond.
Hydrogenation
of Parafin and Alcohol Derivatives of Punicie
Acid--Gas-liquid
chromatography
of the hydrocarbons
isolated
after incubation
of B. Jibrisolvens with cis-9, truns-11 ,cis-13octadecatriene
showed the appearance of a peak not observed in
the hydrocarbon
fraction from a zero time control.
This peak,
amounting
to 21.5% of the hydrocarbon
fraction, exhibited a
retention
time corresponding
to that calculated for an octadecene.
The alcohol derivative of punicic acid also appears to be reduced, since analysis of the reaction products by argentation
thin layer chromatography
showed a spot that corresponded to
truns-11-octadecenol.
Stereospecijcity
of Biohydrogenation
Reaction-Stereospecific
desaturation
of stearic acid by C. vulgaris (15) provided the
rationale by which the stereospecificity
of the reduction of the
cis-9 double bond of cis-9, truns-ll-octadecadienoic
acid was
studied.
In these experiments
cis - 9, truns - 11[9,10 - 3H]octadecadienoic acid was incubated with B. $brisolvens.
The labeled truns-11-octadecenoate
product was isolated as the methyl
ester and converted to stearic acid. Following the addition of
lJ4C-stearic acid, the doubly labeled stearic acid was incubated
with Chlorella.
In each of two experiments, the ratio of 3H:14C
in the oleic and linoleic acids isolated from the algae was the
same as the 3H:14C of the stearic acid substrate (Table I). To
ensure that migration of the labeled hydrogens had not occurred
during incubation
with B. Jibrisolvens, stearic acid containing
only the tritium label was incubated with Chlorella.
The dinitrophenylhydrazone
derivatives of the aldehyde fragments obtained from reductive ozonolysis of the tritiated methyl oleate
were found to contain almost equal amounts of tritium (Table
II).
Another
portion of the labeled oleate was oxidatively
cleaved.
No radioactivity
was observed in either the nonanoic
or monomethyl azelaic acid fragments.
These results show that
the tritium in the cis-9, truns-1119, 10-3H]octadecadienoate
had
remained at positions 9,lO during incubation
with B. fibrisolvens.
Source of Hydrogen in Hydrogenation
Reaction---Initial
attempts to ascertain the source of the reducing hydrogen were
made by incubating
B. jibrisolvens with a series of tritiated

Downloaded from www.jbc.org by guest, on October 8, 2012

as described by Neihaus and Ryhage (23). The monoene fraction from the incubation of punicic acid in D20 was reduced to
the paraffin via the alcohol and mesylate ester (7,8), as previously
described and oxidatively
cleaved by a modified method of
Scheuerbrandt
and Block (24). Since the paraffin was insoluble
in their reaction mixture, the solvent was removed and the
the following solutions were added per 5 mg of unsaturated
hydrocarbon: 0.8 ml of t-butyl alcohol, 0.3 ml of a mixture of 0.02
M Khln04
and 0.19 M NaI04, 0.12 ml of 0.04 M K&Ox, and
finally 0.6 ml of water.
The flask was sealed and stirred for 2
hours at room temperature and the acid fragments were isolated
(24). Mass spectra of their methyl esters were obtained by
using the gas chromatographic
inlet system of a model 9000
LKB mass spectrometer.
A four-foot column of ethylene glycol
succinate-HaPOd
was used with temperature
programming.
Several scans were obtained for all samples and the peaks of
interest were corrected for natural abundance.
Gus-Liquid Chromatography--The
methyl esters of the acids
obtained from incubations with linoleic acid, cr-eleostearic acid,
and the c&runs-conjugated
acid mixture were analyzed by
gas-liquid chromatography.
The paraffins isolated from incubation of B. jibrisolvens with cis-9, truns-11 , cis-13-octadecatriene
An F and M
were also subjected to gas-liquid chromatography.
model 700 flame ionization
instrument equipped with four-foot
columns of 10% diethylene glycol succinate on Chromosorb W
was used.
Other Analytical Procedures-Ester
groups were determined by
the procedure of Snyder and Stephens (25).
Radioactivity
was measured in a Packard Tri-Carb
liquid
scintillation
spectrometer
by usin, 0 a scintillation
solution of
Omnifluor (New England Nuclear) in toluene (4 g per liter).
Infrared spectra were measured in a Beckman IR-8 in carbon
disulfide solution.
Jloleculnr
formulas were determined by accurate mass measurement on a MS-902 mass spectrometer.

and S. B. Tove

5028

Source of Hydrogen
TABLE

and Stereospecificity

of Reduction

II

Vol. 246, No. 16

TABLE

Trilium.
S:orn

in reductive
ozonolysis
fragments
of methyl
oleate isolated
Zhlorella
after incubation
with cis-9, trans-11[9,1
O-aH]octaclecadienoic
acid with Butyrivibrio
jibrisolvens
The oeonide
of methyl
oleate
was reduced
with 2,4-dinitrophenylhydrazine
and the dinitrophenylhydrazones
of nonanal
and methyl
9-oxononanoate
were oxidized
and the water
from
each was collected
and counted.
The specific
activity
of the 9, lodi-3H-cis-9,
trans-11-octadecadienoic
acid was 60 mCi per mmole.
Fragment

Deuterium
cgntent
of fragments
11 ,I$-dimethoxu
octadecanoate
octadecenoate
isolated
after
Butyrivibrio

of methyl
end and carboxyl
end of
prepared
from
methyl
trans-illinoleic
acid incubation
with
$brisolvens

Mass spectra
(70 e.v.) were obtained
with an AEI-12
ter and the peaks of interest
were corrected
for natural

spectromeabundance.

Per cent of parent ions containing

No D atoms

Tritium

1 D atom

2Datoms

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

100.0
80.0

TABLE
TABLE

III

Incorporation
of 3H from 1 -3H-glucose
and VH-glucose
into
11 -octadecenoic
acid and the saturated fatty
acids by
Butyrivibrio
jibrisolvens
Incubations
were
glucose
as described

carried
in the

trans-

out with linoleic

acid and the labeled
text.
The methyl
esters of the satu-

acid were isolated by

Substrate

Saturated acids

Experiment

cis, &s-18:2

IV

(Agn12)

cis, trans,cis-18:3

(Ag.11v13)

1
2
3
1
2

after

incubation

Per cent of parent ions containing

XoD
atoms
15
15
9
7
8

1D
atom
~-__33
16
40
19
24

10

1 13

14

cis-cis-18:2
(A9.12)
cis-trans-&s-18:3
(As.ll.lr)

40
58

in methyl trans-11 -octadecenoate

isolated
of linoleic.acid
and punicic
acid

Substrate

at positions

C@&/j.Hde

7279
366

TABLE
Deuterium

Deuterium
Substrate

trans-11.18: 1

cpm//mde
PH-Glucose
3-3H-Glucose.

of deuterium
in trans-li-octadecene
prepared
from
trans-11 -octadecenoate
product
of linoleic
and punicic
acid incubations
After
isolation,
methyl
trans-11-octadecenoate
was converted
to the paraffin
derivative
and oxidatively
cleaved
to heptanoic
and undecanoic
acids.
The methyl
esters
of these acids were
subjected
to
gas-liquid
chromatography-mass
spectrometer
analyses
on a LKB model 9000 spectrometer
(70 e.v.).
The peaks
of interest
were corrected
for natural
abundance.
The positions
refer to the original
trans-11-octadecenoic
acid, positions
9 and 10
coming
from undecanoic
acid and positions
13 and 14 coming from
heptanoic
acid.

2D
atoms

3D
atoms

4D
atoms

43
60
39
34
36

9
7
3
27
25

0
0
0
13
7

substrates.
No tritium was incorporated
from glucose labeled
in positions 1>2 73 95 9 and 6 or from tritiated succinate or formate.
,Evidence that l-3H-glucose and 3-3H-glucose were metabolized
as expected, i.e. provided reducing equivalents for fatty acid
synthesis, comes from the observation that tritium was found in
the fatty acids synthesized by the cell (Table III).
To determine whether or not water provides the hydrogens
for reduction, incubations
of I?. jlbrisolvens
in DzO were performed.
When incubated in D20, a single deuterium atom was
found to be incorporated
at C-13 during the isomerization
of
linoleic acid to cis-9, truns-11-octadecadienoic
acid (2), but the
hydrogenated
product was not examined.
More recent experi-

ments in which approximately

3 ml of bacterial pellet were
suspended in 12 ml of 99% DzO indicate that, during reduction
of the cis-9, trans-11-octadecadienoic
acid in DzO, 2 additional
atoms of deuterium
were present in the resulting
truns-lloctadecenoic acid (Table IV).
The actual level of deuterium
incorporated
reflects not only the specific activity of the water
but the rate of equilibration
of deuterium with the active hydrogens of the bacterial cell.
Similar experiments were performed with punicic acid as a
substrate.
Since punicic acid does not undergo isomerization
prior to its hydrogenation
to the truns-11-monoene
and since
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.
These experiments analyzed by mass spectrometry indicated
that

deuterium

cis double
tion.

To

was

bond(s)
localize

incorporated

during

the

reduction

but did not reveal the positions

the

incorporated

deuterium,

the

of the

of substitumethyl

ester

of truns-11-monoenoic
acid resulting from linoleic incubation
was converted to the 11,12-dimethoxy
derivative and subjected
to mass spectrometry
(23).
When
treated
in this manner,
the
dimethoxy
compound undergoes cleavage between the methoxyl groups, yielding 2 ions (m/e 129 and m/e 229) corresponding
to the methyl end and the carboxyl end of the methyl truns-lloctadecenoate (23). The results (Table V) indicate that the deuterium atoms incorporated
during hydrogenation
of the A-9,

Downloaded from www.jbc.org by guest, on October 8, 2012

rated fatty acids and trans-11-octadecenoic

silicic
acid-silver
nitrate
column
chromatography.
The fractions emerging
from the column first were taken as saturated
fatty
esters.
Ester
concentrations
were determined
on a portion
of
the sample, and another portion was counted in a liquid scintillation spectrometer.
Radioactivity
measurements
were corrected
for background
and quench. The specific activity of each tritiated glucose
was 100 $Zi per mmole.

VI

Distribution
methyl

Issue of August

I. X. Rosenfeld

25, 1971

DISCUSSION

The hydrogenation
of linolcic acid initially involves the isomerization
of linoleic acid to a cis-9, truns-1 l-octadecadienoic
acid. Several reports on the natire and characteristics of linoleic acid isomerase, the enzyme that catalyzes this reaction,
have been published (2, 3), but, until now, none of the findings
concerning the reduction of the conjugated intermediate
to truns11.octadecenoic
acid have been reported.
As there is no readily available source of this cis-9,truns-lloctadecadienoic
acid intermediate,
punicic acid, cis-9, truns-ll ,
cis-13.octadecatrienoic
acid represents a unique substrate which
facilitates the investigation
of t,he reductive reaction.
It has
been shown (Table IV) that both cis double bonds are hydrogenated, resulting in the same product as that obtained from
linoleic hydrogenation.
It is interesting
to note that, when
ar-eleostearic acid (cis-9, truns-11 , trans.13.octadecatrienoic
acid)
is used as a substrate, no reaction occurs. The inactive aeleostearic acid is a conjugated triene similar to punicic acid
differing only in that the configuration
of the Al3 bond is truns
instead of cis. It is apparent, therefore, that the configuration
of the conjugated truns double bond system imparts a degree of
alteration to the molecule such that the organism is incapable of
reducing the cis bond of the conjugated triene.
The similarity
of deuterium
distribution
at both cis bonds
indicates that, unlike linoleic acid isomerase, the carboxyl group
is a dispensable feature of the substrate.
Preliminary
experiments showed that the alcohol and paraffin derivatives of punicic

5029

acid were hydrogenated

and, thus, support this conjecture.
Some naturally occurring compounds that contain a truns-conjugated double bond system, such as the carotenes, escape
hydrogenation
in the rumen (27). The findings with cu-eleostearic acid and the punicic acid derivatives suggest that it is
the presence of the truns configuration
rather than the absence
of a carboxyl group which accounts for their lack of hydrogenation.
Experiments with tritiated glucose (labeled in positions 1, 2, 3,
5, and 6) showed that the hydrogen that reduces the double bond
did not come directly from glucose. Similarly, absence of tritium
incorporation
in truns-11-octadecenoic
acid from labeled formate
and succinate, as well as the absence of deuterium incorporation
from a totally deuterated algal hydrolysate, indicated that the
direct addition of hydrogen from an organic substrate was unlikely.
In contrast, the fact that 2 deuterium atoms were incorporated from D,O during the hydrogenation
of cis-9, truns-lloctadecadienoic
acid and 4 deuterium
atoms from DzO were
incorporated
in the biohydrogenation
of punicic acid indicates
that water is the immediate source of hydrogens used to reduce
the cis bond(s).
These results, however, do not preclude the
direct reduction of a carrier by an organic substrate if the hydrogen carrier can undergo rapid exchange with water.
Examination
of the isotope distribution
in the reduced products
showed that the position(s) adjacent to the truns double bond
contains less deuterium
than the distal position(s).
This distribution indicates that discrimination
against deuterium had occurred at C-10 of cis-9,truns-ll-octadecadienoic
acid, and at (-10
and C-13 of punicic acid. Therefore, the hydrogens added at
C-10 or C-13 must have experienced at least one more bondbreaking event than those added at C-9 or C-14. These results
lead to the suggestion that the mechanism of biohydrogenation
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
finally completed by a hydride ion provided by an unknown carrier.
Since ferredoxin occurs commonly in anaerobic organisms, one
might expect this electron carrier to be involved in biohydrogenation. However, we were unable to observe a ferredoxin band on
a DEAE-cellulose
column following chromatography
(28) of cell
extracts of B. $brisolvens.
Upon biohydrogenation
and reduction of the monoenoic acid to
stearic acid, it is possible, with the stearic acid as a substrate for
Chlorellu, to determine the stereochemistry
of hydrogen addition
by B. jibrisolvens.
Morris has used this approach to study the
stereospecificity of the biohydrogenation
of oleic and elaidic acids
by mixed rumen flora (29), and Schroepfer, using Corynebucterium
diphtheriue instead of ChZoreZZu, determined the stereospecificity
of the hydroxylation
of oleic acid (30). As reported in the previous paper of this series, the same approach was used to show
the stereospecificity of hydrogen addition at C-13 of linoleic acid
during its isomerization
(31).
If the biohydrogenation
of cis-9, truns-11-octadecadienoic
acid
occurs by cis addition, then either DD or LL-9, 10-di-aH-truns-lloctadecenoic acid would result.
Desaturation
by Chore&x of the
stearic acid derived from the truns-ll-octadecenoic
acid would
yield oleic and linoleic acids showing either complete recovery of
the tritium for the D-labeled enantiomer or complete loss of tritium for the L-labeled enantiomer.
The truns addition of hydrogen by B. fibrisolvens would yield threo-di-aH-truns-ll-octudcccnoic acid, and the oleic acid isolated from Chlorellu would be

Downloaded from www.jbc.org by guest, on October 8, 2012

trans-11-octadecadienoic
acid were located in the carboxyl portion of the molecule.
The distinct positions of substitution
were obtained by reducing the deutcrated truns-11-octadecenoic
acid from the punicic
acid and linoltic acid incubations
to the trans-11-octadecene.
Oxidative cleavage and mass spectrometry
of the methyl esters
of the heptanoic acid and undecanoic acid fragments allowed
the use of the McLafferty
rearrangement
to determine
the
location of deuterium in the original truns-1 l-octadecenoic
acid.
The major peak of methyl esters longer than Cs is due to a
rearranged ion of m/e 74. This ion contains 3 hydrogen atoms:
2 from the a-carbon and 1 from the y-carbon of the fatty acid
methyl ester (26). In this case, the two hydrogens bonded to
the a-carbon of the methyl heptanoate fragment represent the
hydrogen atoms at C-13 of the truns-11-octadecenoic
acid.
Those bonded at C-10 of the trans-11-octadecenoic
acid would
correspond to a-hydrogens of the methyl undecanoate fragment.
The appearance of a large peak at m/e 75 in the spectrum of
each of the monocarboxylic
methyl esters from both substrates
indicates that hydrogen from HZ0 is incorporated
at C-10 of
linoleic acid and at C-10 and C-13 of punicic acid.
From the ratio of the m/e 74 ion to the m/e 75 ion and the
assumption that all of the deuterium incorporated
was bonded
to the carbons of the cis double bond(s), the distribution
of
deuterium at each of the positions of the double bond could be
calculated.
The results (Table VI) show that the carbons adjacent to the truns double bond contain less deuterium than those
From the mass peaks associated with
distal to the truns bond.
the parent ions, it may be calculated that 3056 of the hy-drogen
atoms at C-13 and C-14 and 28T1 of the hydrogen at C-9 and
These results, together with
C-10 were replaced by deuterium.
the similarity in distribution,
suggest that both of the cis bonds
of punicic acid were hydrogenated by the same system.

and S. B. Tove

5030

Source of Hydrogen

and SkreospeciJicity

expected to show one-half of the tritium label. The results (Table I) showed complete recovery, and reductive and oxidative
ozonolysis of the oleic acid showed that the tritium label had not
moved during incubation.
We conclude, therefore, that the biohydrogenation
of L-9, trns-1 I-octadecadienoic
acid by B.
fibrisolvens
occurs by cis addition to the D side of carbons 9 and 10.
Morris (29) has shown that the biohydrogenation
of oleic acid
is
involves cis addition to the L side. However, B. fibrisolvens
unable to hydrogenate oleic acid (32). Consequently, although
the biohydrogenation
of oleic and &s-9, trans-11-octadecadienoic
acids is similar in that both involve cis addition to a cis double
bond, it is clear that, the two systems are different.
Studies with a cell-free system capable of carrying out biohyParticular effort is being directed
drogenation
are in progress.
toward the elucidation
of the nature of the electron donor and
carrier.
Acknowledgments-We

wish

to thank

Dr.

Marion

Miles

of the

Department
of Chemistry for some of the mass spectrometric
analyses. We also thank Drs. D. P. Schwartz and 0. W. Parks
of the USDA, Washington,
D. C., for helping us separate the
is extended
trometry.

derivatives

and further

to Dr. Parks for his help in gas-liquid

We are also indebted

to Dr.

L. J. Morris

appreciation

mass specfor his many

helpful comments and discussions.

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2,4-dinitrophenylhydrazone

of Reduction