Hydrogen peroxide: A potent activator of dioxygenase activity of soybean lipoxygenase

vol.

166,

No.

January

BIOCHEMICAL

1, 1990

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

15, 1990

HYDROGEN PEROXIDE

Received

ACTIVITY

OF

Ashoke Mitra, Jayanta Chaudhuri, P. Kulkarni, and Ira Richards Janus2 2. Byczkowski,

Arun

Florida

: A POTENT ACTIVATOR OF DIOXYGENASE SOYBEAN LIPOXYGENASE

417-423

Toxicology Research Center, College of Public Health, MHH-104, University of South Florida 13301 Bruce B. Downs Blvd., Tampa, FL 33612

October

23,

1989

Hydrogen peroxide, an ubiquitous biologically occurring peroxide, was found to stimulate the dioxygenase activity of soybean lipoxygenase at the physiologically attainable concentration. The increase in enzyme specific activity was directly proportional to hydrogen peroxide concentration up to 0.5 nM. A decrease in the stimulation of dioxygenase activity was observed at higher concentrations. At low enzyme concentration up to 28-fold stimulation was noted when the formation of lipid hydroperoxide was monitored spectrophotometrically. The stimulation was further confirmed by increased oxygen uptake. It is proposed that the mechanism for --in vivo activation involves hydrogen peroxide. 0 1990AcademicPress, Inc.

Lipoxygenase containing

has

reported

and

the

Of

of

hydroxy products

acids, involving

pathophysiological

can

lag

be avoided

the

biomedical of recent

lipoxins, lipoxygenase

by the

In

acid

years.

in initial

the

are

studies dioxygenation

addition

417

synthase view

of

the

of

end is

The generation

of

as

hydroperoxy

leukotoxins

implicated

in

and a variety

inflammation have reaction

Copyright 0 I990 rights of reproduciion

and

shown

of hydroperoxide

Afl

enzyme

oxygenation

such

as

iron

the

importance

fatty

such

Several

non-heme

protein

leukotrienes,

conditions

exists

a

leukotriene

and end products

(4,5). phase

(l),

activities.

in

is

a single

(3)

process

intermediates

hypersensitivity kinetic

of

attention

fatty

being

dioxygenase

enzymatic

greater

plethora

other

have

realization

receiving

or

to

hydroperoxidase

increasing products,

EC 1.13.11.12)

Despite

enzyme.

been

(2) r

(lipoxidase,

that

(6-S) to

a that the

0006-29 1X/90 $1 SO by Academic Press, Inc. itr ap jbrm reserved.

Vol.

166, No. 1, 1990

reaction

mixture

Funk

--et

(9)

reported

process.

phase,

the the

AND BIOPHYSICAL

or by increasing

al.

saturable

and

BIOCHEMICAL

It

protein

that

enzyme

activation

is generally

exhibits

observed

the

RESEARCH COMMUNICATIONS

concentration of

assumed

full

dioxygenation

rate

dioxygenase that

expression

of is

(6,9). is

after

the

enzyme

taken

a lag

activity

as

maximal

velocity. Hydrogen constantly living

peroxide, formed

cells.

ineffective lipoxygeanse peroxide xenobiotics

during

Earlier a

via

lipid the

(3) * were

was thought

important activate

of

that

Since,

to

the

reinvestigate

dioxygenase

in

peroxide

that

the

the

whather activity

of

hydrogen

of and

of soybean

peroxidase

same protein, hydrogen

the

is of

co-oxidation

activity

with

in

activity

dioxygenase

be associated

MATERIALS

hydrogen

was demonstrated

hydroperoxide

is

reactions

dioxygenase

hydroperoxidase

shown to

the

it

product,

biochemical

indicated

Recently

can replace

metabolic

normal

stimulator

(6,lO).

activities

can also

various reports

as

lipoxygenase

an ubiquitous

it

peroxide

enzyme.

AND METHODS

Soybean lipoxygenase-Type V (737,000 Sigma Units/mg protein; M.'W.=lOS kdaltons), linoleic acid (99% pure) and 30% were purchased from Sigma Chemical Co., St. hydrogen peroxide Louis, MO. Dioxygenase activity of lipoxygenase was measured either by spectrophotometric oxygen uptake or spectrophotometrically. For assay, reaction medium (3.0 ml final volume) in the sample contained indicated concentration of enzyme (0.05-0.5 cuvette prepared linoleic acid (0 - 100 uM), hydrogen nW , freshly The reference peroxide (O-3.0 nM) and Tris buffer, pH 9.0. cuvette contained linoleic acid + hydrogen peroxide in 50 mM Tris All spectral measurements were done using a buffer, pH 9.0. Gilford Response UV-VIS spectrophotometer at room temperature in Increase in absorbance at 234 nm was open quartz cuvettes. monitored and an extinction coefficient of 25 mM'l cm-' was used for quantitation of hydroperoxylinoleic acid. Oxygen uptake was electrode on a YSI Model 5300 biological monitored by a Clark oxygen monitor under identical assay conditions except that final the reaction mixture was 2.0 ml. Measurements were volume of begun immediately after the addition of enzyme. Linoleic acid was prepared according to the method of Tappel with some modification. Stated briefly, to a et a& (11) ethanol, mixture of 50 ul of linoleic acid and 50 ul of absolute 10 ml of 50 mM Tris buffer pH 9.0 (deaerated with nitrogen for 10 mixture was minutes) and 1 drop of Tween 80 were added and the 418

Vol.

166,

No.

vortexed into a substrate linoleic

1, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

for 5 seconds. The preparation was immediately poured cold beaker and maintained on ice. This resulted in a solution without spectrophotometrically detectable acid hydroperoxide. RESULTS

Two effects

is

assay

hydrogen

of

soybean it

independent

lipoxygenase. evident

that

formation

alone.

evident

the

that

increase results measured

the

From

spectral

in

suggested

that

reaction

during

the

increase

the

reaction

than

that

given

in

in

Fig.

200

220

240 Wavelength

Fiqure (0.15

1. nM)

Linoleic in the

it

212

enzyme

is

clearly

a

marked these

was

being

(Fig.la)

234 nm turbidity

was of

of the

the

products

be established.

a

o.o-

with

together,

identity

chemical to

lb

at

product

caused

the

la,

at

the

reaction

as a change

Fig.

peak

observed

Taken

at

of

absorbance acid

peroxide

absorbance

remains

in

peroxide

uptake.

However,

given

linoleic

hydrogen

the

activity

in

dioxygenation in

such

etc.

data

of hydrogen oxygen

artifacts,

mixture

data

assess

to

dioxygenase

the of

From the

employed

increase

higher

addition

and the

the

presence

(14-fold)

due to

formed

on the

was significantly

and substrate

not

peroxide

Concomitantly,

In

decreased.

were

a time-dependent

234 nm was observed. nm

methods

b

260

260

5001

300

0

15 Time

(nm)

acid presence

30

45

60

(seconds)

(70 uM) metabolism by soybean lipoxygenase or absence of hydrogen peroxide (0.5 nM). (a)Spectral analysis: [A] linoleic acid: [B] linoleic acid + enzyme + hydrogen peroxide after 1 min.; [C] linoleic acid + enzyme after [D]: linoleic acid + enzyme + hydrogen peroxide after 3 min. 3 min.: (b) Oxygen uptake (ng atoms/min) in the presence or absence of hydrogen peroxide.

419

Vol. 166, No. 1, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a

0, 0

Figure

b

’ r 1 2 3 Hydrogen peroxide (nM)

2. Dioxygenase

4

activity

25 50 75 Linoleic acid (PM)

0

of soybean

lipoxygenase

100

(0.15

nM)

in the presence of (a) 70 uM of linoleic acid and indicated concentration of hydrogen peroxide: (b) 0.5 nM hydrogen peroxide and indicated concentration of linoleic acid. The spectrophotometric assays were carried out as described in Materials and Methods.

The effect

results of

in

in

Fig.

maximal

activity

lower

hydrogen (data

of

indicated stimulation

of

increase

Further

increase in

The data stimulation

presented

(28 to

Further

an

of

in

extent

the

acid the

in

in

the

of

protein). 420

increase

of

inhibition of

reaction

0.5

nM

in The

dioxygenase

the data

activity

was increased

up

to

(70-100

stimulation.

1 indicate

7 fold

3 nM) resulted

effects

that

proportional

approximately

that

dioxygenase

concentration

of the

stimulation

are

concentration.

substrate

Table

peroxide

enzyme

dioxygenase

concentration

decline

the

in

stimulation.

acid

inversely fold

20-fold

resulted

evaluate

concentration

(up to

linoleic

a gradual

was

concentration

about

rates

in

hydrogen

peroxide

represents

on the

to

concentrations

enzyme

when linoleic

uM) resulted

compared

The hydrogen

Fig.2b

different

an

of

concentration

shown).

presence

uM.

of

peroxide

peroxide

70

2a.

nM. Higher

hydrogen

designed

concentrations

extent

not

experiments

stimulation

was 0.5

a

the

increasing

presented caused

of

with

the to

0.05

stimulation

magnitude the nM with

of

enzyme enzyme

as

0.5

nM

Vol.

166,

No.

1,

BIOCHEMICAL

1990

Table

1.

Stimulation

AND

of soybean

Enzyme

BIOPHYSICAL

Ilpoxygenase

RESEARCH

by hydrogen

"202

Dloxygenase

(0.5 nM)

Activity’

0.05 0.10

+

64.16 2.76

26

0.10

+

56.64

21

W

COMMUNICATIONS

paroxlde

Fold Stlmulatlon

2.32

0.05

0.15

2.24

0.15

+

44.64 2.12

20

0.20

+

36.20 2.00

19

0.30 0.30

+

27.17

14

0.20

0.50

2.46

0.50

+

16.56

7

‘Specific activity is expressed in f.tLroles of linoleic acid hydroperoxide formed/minlnmole enzyme. The spectrophotometric assays were carried out in the presence of 70 )LM linoleic acid as described in Materials and Methods.

DISCUSSION Although understand

the

dioxygenase

activity

of

peroxide

phenomenon

et

was

effective

al.

and

activators

and

than

that and

stimulate

of other

t-butyl

linoleic

40,

13-linoleic

acid

enzyme to

which

whereas

Haining

are

reduce 60,

acid

activity

the

of

Earlier

were

(13,14). 421

(6)

the

cumene as

They lag

addition,

also

only

ineffective

and by a mechanism

have

acid

dioxygenase

period.

In

de

hydroperoxide

for

and 80 reduced

factors

of

peroxide,

lag

to

specificity

and Axelrod

substrates

hydroperoxide.

past

9-linoleic

hydrogen

concentration

endogenous

dioxygenase

that

question

hydroperoxide not

Tween 20,

at much higher

only,

ATP

that

the activation

resolved.

whereas,

could

in

being

According

activators

hydroperoxide

made

the

from

the

of compounds

as

reported

far

activating

was not.

hydroperoxides

been

lipoxygenase,

observed

in

have

of hydroperoxide-caused

remained

(12)

hydroperoxide

serve

of

has

Groot

attempts

many

been

also

period

but

different calcium, shown

to

Vol.

166, No. 1, 1990

This

BIOCHEMICAL

is,

stimulation

of

our

knowledge

dioxygenase

physiologically spite

to

attainable

of

the

AND BIOPHYSICAL

activity

of

active

acccumulate

this

example,

steady

concentration

1.0

nM-100 results

Our inactivation

of

peroxide

(6,10,16).

indicates

that

actually

from

with

(i)

were

But rates

found

peroxide

with

(data

shown).

was reported linoleic

enzyme

in

66,666

fold

molar

peroxide

acid

in

excess our

basis, is

1.33:1;

occur;

those

in

results

50,000

hydroperoxide

in

2a)

times

Apparently,

of

seems

to

in view

of

linoleic should

acid increase

furthermore,

the

acid

presence

of

alone hydrogen

hydroperoxide-catalyzed process

acid (9).

requires

that (0.15

nM

hydroperoxide, In

contrast

to

of i.e.

this,

on

ratio

of

enzyme:hydrogen

indicate

that

hydrogen

peroxide

effective

dioxygenase the

The mechanism

(molar

more

stimulating

also to

concentration

enzyme)

is

linoleic

acid

10 uM linoleic

study

difficult

to

(ii)

the

hydrogen

peroxide

peroxide

autooxidized

Linoleic

Fig.

approximately

lipoxygenase.

not

of the

is

velocity

to be a saturable

of

it

due

on

this

hydroperoxide is

hydroperoxide

presence

in

hydroperoxide.

reaction

than

of

hydroperoxide

acid

does

reports

hydrogen

since

activation

ranging

concentrations

Whether

linoleic

extensively

not

earlier

by hydrogen

the

to be lower

activation high

this

the

activity

the

then

its

For

(15).

gathered

at present of

of if

alone

time.

initial

free

that

following:

hydroperoxide

a

be answered

liver

acid

some

peroxide

subnanomolar

or linoleic

In

peroxide.

concentrations

at

by

peroxidases,

for

evidence

peroxide

dioxygenase

be different

the

enzyme

acid

of

by high

as an activator.

the

linoleic

activation

enzyme

with

the

peroxide.

generated

reported

agreement

However,

cannot

prepare

the

the

serve

activates

required,

in

hydrogen

can

alone

are

and

of hydrogen

been

on

lipoxygenase

of hydrogen

endogenously

nM have

report

of

catalase

tissues

between

first

concentration

presence

state

the

RESEARCH COMMUNICATIONS

observed 422

than activity phenomenon

is

linoleic

acid

of

soybean

of

hydrogen

Vol.

166, No. 1, 1990

BIOCHEMICAL

peroxide-caused uniquely the

restricted

mammalian

response

was

(details would vivo

activation to enzymes observed

to be published like activation

to

propose of

AND BIOPHYSICAL

of

dioxygenase

soybean

enzyme

as

well,

since,

with

rat

the

liver

elsewhere). hydrogen

RESEARCH COMMUNICATIONS

activity alone similar and

In view

peroxide

but

as the

not

extends

to

synergistic

lung of

is

these trigger

lipoxygenase findings for

we the

-in

lipoxygenases. ACKNOWLEDGMENTS

This study was supported for Tobacco Research USA Inc., Son, Inc.

in part by a grant from The Council and by a gift from S.C. Johnson &

REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Chem. 242, Hamberg, M. and Samuelsson, B. (1967) J. Biol. 5329-5335. Kaneko, S., Ueda, N., Tonai, T., Maruyama, T., Yoshimoto, T. and Yamamoto, S. (1987) J. Biol. Chem. 262, 6741-6745. Kulkarni, A.P. and Cook D.C. (1988) Biochem. Biophys. Res. Commun. 155(2), 1075-1081. Samuelsson, B. (1983) Science 220, 568-575. Malle, E., Leis, H.J., Karadi, I. and Kostner, C.M. (1987) Int. J; Biochem. 19, 1013-1022. Haining, J.L. and Axelrod, B. (1958) J. Biol. Chem. 232, 193-202. W.L. and Lands, W.E.M. (1972) J. Biol. Chem. 247, Smith, 1038-1047. Tappel, A.L., Boyer, P.D. and Lundberg, W.O. (1952) J. Biol. Chem. 199, 267-281. Funk, M.O., Kim, S.H-S. and Alteneder, A.W. (1981) Biochem. Biophys. Res. Conunun. 98(4), 922-929. Rouzer, C.A. and Samuelsson, B. (1986) FEBS Lett. 204(2), 293-296. Tappel, A.L., Boyer, P.D. and Lundberg, W.O. (1953) Arch. Biochem. Biophys. 42, 293-304. deGroot, J.J.M.C., Garssen, G.J., Veldnik, G.A., Vliegenthart, J.F.G. and Boldingh, J. (1975) FEBS Lett. 56(l), 50-54. Rouzer, C.A., Shimizu, T. and Samuelsson, B. (1985) Proc. Natl. Acad. Sci. USA 82, 7505-7509. Rouzer, C.A. and Samuelsson, B. (1985) Proc. Natl. Acad. Sci. USA 82, 6040-6044. Oshino, N., Britton, C., Sies, H. and Bucher, T. (1973) Arch. Biochem. Biophys. 154, 117-131. Mitsuda, H., Yasumoto, K. and Yamamoto, A. (1967) Agric. Biol. Chem. 31(7), 853-860.

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