Double dioxygenation of arachidonic acid by soybean lipoxygenase-1

BIOCHEMICAL

Vol. 74, No. 3, 1977

AND BIOPHYSICAL

DOUBLE DIOXYGENATION

Gary

S. Bild,

OF ARACHIDONIC ACID BY SOYBEAN

LIPOXYGENASE-l* ** S. Ramadoss , Sanghee

Candadai

Department

Received

December

RESEARCH COMMUNICATIONS

Lim and Bernard

***

Axelrod

of Biochemistry

Purdue

University

West Lafayette,

Indiana

47907

15,1976

SUMMARY: Soybean lipoxygenase-1, has been found to catalyze the incorporation of two oxygen molecules into arachidonic acid. The product appears to be 8,15 dihydroperoxy-5,9,11,13-eicosatetraenoic acid. This is apparently the first report of the enzymatic production of a conjugated aliphatic triene in vitro. Lipoxygenase been

reported

ular

oxygen

adiene

is

to be in thrombocytes to polyunsaturated

system.

Soybeans

One of these,

(2).

an enzyme which

peroxy

isomer

oxygen

only

from

(1).

fatty

contain

lipoxygenase-1, linoleic

identification

arachidonic

acid.

concentrations, a reaction

isoenzymic

of arachidonic acid

By utilizing

consumes

containing

(3). product

lipoxygenase-1 acid

the

Lipoxygenase

of a new reaction

arachidonic which

acids

catalyzes

catalyzes

acid.

to the w-6 position

in many plants It

several

peroxy-5,8,11,13-eicosatetraenoic the

occurs

is

converted

two molecules MATERIALS

and has recently

the addition

the cis-cis-1,4-pentforms

of

formation

the enzyme of the

has been reported acid This

to produce

at relatively a conjugated

13-hydroto add

15-hydro-

communication

of lipoxygenase-1

into

of molec-

reports acting high

on

enzyme triene

in

of oxygen. AND METHODS

Chemicals - All fatty acids (99% purity) were Inc. N-Methyl-N nitroso-p-toluenesulfonamide Chemical Company. N,N-Dimethyl-p-phenylenediamine x

obtained from NuCheck Prep, was purchased from Aldrich HCl was obtained from

Journal Paper No. 6557, Purdue Agricultural Experiment Station. Supported by NSF Grant BMS 74-13883. ** Present address: Forschungsstelle Vennesland der Max-Planck-Gesellschaft, Berlin 33, Harnackstrasse 23, Germany. *** To whom reprint request may be addressed. Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

949

Vol. 74, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL

Sigma Chemical Company. Thin layer chromatography gel G, were supplied by Analtech Inc.

RESEARCH COMMUNICATIONS

(TLC)

plates

of silica

Enzyme Purification and Assay - Lipoxygenase-1 which was prepared by the method of Christopher et al. (4) was used throughout this work. The enzyme was purified from Amsoy soybeans by ammonium sulfate fractionation, chromatography on DEAB-Sephadex and chromatography on hydroxyapatite. The enzyme preparation, specific activity of 180 units per mg protein, was stored as an ammonium sulfate precipitate. The enzyme was diluted in 0.2 E sodium phosphate buffer pH 6.8 before use. One enzyme unit represents the consumption of 1 pmole of oxygen per minute at 15'C with linoleic acid as substrate at pH 9.0 as defined by Christopher Activities were determined --et al. (4). using a Clark oxygen electrode (Yellow Springs) in a Gilson Medical Oxygraph, model KM. Preparation of Di-dioxygenated Product - A suspension of arachidonic acid was prepared by stirring 400 mg of the fatty acid with a small amount of water and 0.35 ml of Tween 20 and then continuing to mix while adding small increments of water until a volume of 10 ml was obtained. The suspension was made neutral with 2 N NaOH and the volume brought to 100 ml with distilled water. The reaction mixture used to obtain the product contained 180 ml of 0.2 g sodium phosphate buffer, pH 6.8, 1330 enzyme units and 10 ml of the above -4 ubstrate mixture to give a final concentration of arachidonate of 6.65 x 10 M. Oxygen was bubbled through the reaction mixture at room temperature with constant stirring. After 10 minutes, a small aliquot of the reaction mixture was scanned in a spectrophotometer to confirm complete conversion of conjugated diene to conjugated triene. The incubation mixture was immediately reduced with dissolved in 10 ml of ethanol. The reduction was allowed 1 hour at room temperature with constant stirring.

200 mg of SnC12 to proceed for

The reaction mixture was extracted twice with 2 volumes of ethyl acetate. The combined extract from 10 runs was evaporated to dryness under vacuum. The residue was taken up in 10% ether in hexane and loaded onto a SilicAR CC-4 column. The column was eluted successively with 200 ml portions of the following ether-hexane mixtures: 10:90, 20:80, 50:50 and finally pure ether. Only the 100% ether eluate contained significant amounts of the product. The fraction was evaporated to dryness under vacuum. The residue was spotted on 1000~ silica gel G TLC plates and developed in chloroform-methanol-acetic acid-water (90:8:1:0.8, v/v/v/v>. Material with an Rf of 0.60, visualized as a dark band under ultraviolet light, was scraped from the plate. This product was extracted in chloroform-methanol (2:1, v/v) and rerun on a second 1000 u plate. The material was methylated in ether (10:90, v/v> for 1 hour. The chloroform-methanol (2:1, v/v) and as before. One major spot appeared was used for mass spectral analysis.

the dark with diazomethane in methanolmethylated material was taken up in spotted on a 250 u TLC plate and developed This substance with an Rf value of 0.77.

Analysis of the product - A Perkin-Elmer Model 467 grating infra-red spectrophotometer employing a KBr cell and chloroform as solvent was used for infrared analysis. NMR analysis was carried out on a Varian T-60A spectrometer. Mass spectra were taken at 19O'C on a CEC 21-110 double focusing high resolution spectrometer.

950

BIOCHEMICAL

Vol. 74, No. 3, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MINUTES

Fig. 1 Oxygraph recording of 02 consumption in a reaction mixture containing 8.21 units/m140f lipoxygenase and a final concentration of arachidonic acid of 1.18 x 10 E in 0.z sodium phosphate buffer pH 6.8 at 15'C. Total volume of reaction mixture was 1.50 ml.

RESULTS AND DISCUSSION Measurement

of oxygen

of lipoxygenase-1 indicated

that

and that

another

20 minutes

with

(Figure

slowly ure

trienes, arachidonic

This as,

while

triplet for acid

(5),

than

during

appeared

new peaks to those

obtained

by the alkaline

or as seen in a- or

951

H-eleostearic

with

at 15°C 1 minute

in a scanning

to conjugated

the diene (260,

units

the next

out

at 238 run corresponding

three

obtained

Oxygraph

was carried

continued,

that

of 12.3

in less

was consumed

same reaction peak

reaction

was consumed

As the reaction

corresponds

example,

the

at pH 6.8 in the

of oxygen

When the

rapidly.

disappeared

2).

of oxygen

an absorbance

appeared

during

acid

equivalent

1).

spectrophotometer diene

arachidonic

1 equivalent 0.85

consumption

269, other

peak

nm)

279 nm) (Figconjugated

isomerization acids

(238

(6).

of Therefore

Vol. 74, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL

I

RESEARCH COMMUNICATIONS

20 min

0.60

0.5 min 0.20

-

; I 220

0.00

yy 260

240

260

300

nm Fig. 2 Spectrophotometric sca3 of a reaction mixture containing (5.67 units/ml) and 4.95 x 10 g arachidonic acid in 0.2PJ sodium buffer pH 6.8 at room temperature. Total volume was 3.0 ml.

it

appears

that

one molecule as in the

under

of oxygen normal

Infrared

rate

the presence

absorptions

at 1040 is

of a trans

ethylenic

indicative

hydroxyl

group

hydroxyl

peak was not

Mass spectral

(secondary

triene

hydroxyl)

proton, seen after

analysis

4.2

of the methylated

952

of oxygen

diene

to a conjugated

triene.

after

SnC12 reduction

clearly

(968 cm-')

acid

ppm; hydroxyl

adds

diene,

molecule

and at 3540 cm

fatty

the sample

rapidly

a conjugated

a second

absorption

of an unsaturated

(methine

to produce

the conjugated conjugated

-1

lipoxygenase-1

and adds

of the

cm

spectrum

acid

reaction,

converting

analysis

specified,

to arachidonic

lipoxygenase

at a much slower

showed

the conditions

lipoxygenase phosphate

-1

possessing proton,

was treated material

and alcoholic The NMR

.

a secondary

6.3 ppm). with produced

The

D20. from

arachi-

BIOCHEMICAL

Vol. 74, No. 3, 1977

donic

acid

compound peaks

revealed containing

appeared

loss

a molecular 4 double

groups

Thus

at

TLC plates diamine

loss

[C21H3203,

of C8H1302]

product

with

the

insertion

The somewhat

groups.

10~s

Other

~~01,

0f

to a C-21

301

of H20 and C5Hlll,

and 191 [M-(141+18); analysis

is

major [M-(18+31);

209.157

loss

compatible

presence

imprecise

of H20 and

with

two hydroxyl

ion

molecule

slower

by lipoxygenase-1

is

well-known

presented

groups

of oxygen

to have we propose

dihydroperoxy-5,9,11,13

test

on

N,N-dimethyl-p-phenylene test

in the presence

The normal

rate.

gave a positive

quantitative

of 2.5 hydroperoxide

second

From the data

reaction reagent,

of ferrous

at a considerably

series

loss

the peroxide-specific

of the

8,15

M-18;

of the enzymatic

on the oxidation

dicated

corresponds

and two hydroxyl

the mass spectral

HCl (7).

based

also

of

for

hydroperoxides

thiocyanate

per molecule occurs

in-

(8).

The

at pH 9.0,

although

(monohydroperoxidation)

a pH optimum that

of about

arachidonic

eicosatetraenoic

reaction 9.0.

acid

acid

is

converted

by the following

of reactions:

The IR spectrum analogy

clearly

to the known

we tentatively while

to be established rapidly

at m/e 350 which

the C-8 and C-15 positions.

The final

uration

bonds

at m/e 332.235

M-141;

C8H13021.

(9))

ion

of H20 and OCH31, 261 [M-(18+71);

[C13H2102,

into

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

inserted

the

indicated structure

assume 11 is

the

of trans

of the product that

obtained

the 9 and 13 bonds

of the -cis

by direct into

the presence

configuration,

investigation.

from

are of

953

the

trans

this

oxygen

as has been shown

bonds.

linoleic

although

The first

w-6 position,

double

By acid config-

remains

molecule

is

by Hamberg

and

BIOCHEMICAL

Vol. 74, No. 3, 1977

Samuelsson of oxygen triene

(3).

arachidonic

can be inserted

system.

This

open conjugated lipoxygenase

With

triene acting

AND BIOPHYSICAL

acid

it

in the w-13

is

the

first

and also as a double

appears

position,

report appears

of the

RESEARCH COMMUNICATIONS

that thus

a second producing

enzymatic

to be the first

molecule a conjugated

formation

of an

observation

of

dioxygenase.

ACKNOWLEDGEMENTS The authors

wish

mass spectrometer for

their

assistance

to thank

analysis with

Dr.

and Dr. the

R. G. Cooks for J. Wolinsky,

Paul

his

assistance Pilch

in

the

and Joe Valko

NMR analysis. REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9.

Nugteren, D. H ., (1975) Biochem. Biophys. Acta, 380, 299-307. E. K., and Axelrod, B., (1972) Biochem. Christopher, J. P., Pistorius, Biophys; Acta, 284, 54-62. Hamberg, M., and Samuelsson, B., (1967) J. Biol. Chem. 242, 5329-5335. Christopher, J., Pistorius, E., and Axelrod, B., (1970) Biochem. Biophys. Acta, 198, 12-19. Glick, D. (1957) Methods of Biochemical Analysis, pp. 99-138, Interscience Publishers, Inc., New York. D. H. (1953) J. Am. Oil Chem. Paschke, R. F., Tolberg, W. and Wheeler, sot., 30, 97-99. of Organic Johnson, R. M. and Siddiqui, I. W., (1970) The Determination Peroxides, pp. 52-53, Pergamon Press, Oxford. Koch, R. B., Stern, B., and Ferrari, C. G., (1958) Arch. Biochem. Bio78, 165-179. @vs., Privett, 0. S., Nickell, C., Lundberg, W. O., and Boyer, P. D., (1955) J. Am. Oil Chem. Sot., 2, 505-511.

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