Singlet oxygen formation during hemoprotein catalyzed lipid peroxide decomposition

BIOCHEMICAL

Vol. 76, No. 2, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SINGLET OXYGEN FORMATION DURING HEMOPROTEIN CATALYZED LIPID Fred

J.

Hawco,

Cindy

R. O'Brien

and Peter

PEROXIDE DECOMPOSITION

J. O'Brien

Department of Biochemistry Memorial University of Newfoundland St. John's, Newfoundland, Canada AlC 5S7 Received

April

1,1977

Summary The singlet oxygen trap diphenylfuran was rapidly oxidized to cis dibenzoylethylene during the decomposition of linoleic acid hydroperoxide catalyzed by ceric ions, methemoglobin or hematin. This conversion was enhanced in a deuterated medium and inhibited by other singlet oxygen quenchers or traps. The chemiluminescence accompanying the decomposition of the linoleic acid hydroperoxide was also markedly enhanced in a deuterated medium and inhibited by other singlet oxygen quenchers or traps. Antioxidants markedly inhibited these reactions. It is concluded that singlet oxygen is formed in substantial quantities during the metal catalyzed decomposition of linoleic acid hydroperoxide. Singlet

molecular

oxygen

or by microwave

discharge

polysaccharides

and nucleic

lipid

peroxidation

(2).

Its

(1).

formation

biological

(3)

its

formation

but

this

oxygen (8).

by others

oxidation

formation

Chemiluminescence,

of linoleic different Abbreviations:

bases

has also

Evidence

to cooxygenate

('02)

It

shown

acid from

involving recently

hydroperoxide the dimol

to modify

acids,

systems

could

therefore

for

formation

its

refuted

(4).

photooxygenation fatty

acids

in enzyme inactivation to be cytotoxic

cells

be of considerable

investigators lipid

and

and

to tumour

by lipoxygenase

Other

microsomal

has been have suggested

peroxidation

system

(5,6)

(7). hydroperoxide oxygen self

traps

reported by ceric

LAHPO, Linoleic acid DPF, Diphenylfuran; 1,4 Diazobicyclo[2.2.2]

in benzene (8).

reaction

A mechanism

of peroxy be emitted

during

ions

was found

to have

reported

hydroperoxide; TLC, Thin octane.

for

layer

has previously for

radicals

to

chemiluminescence

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

amino

shown

of set-butyl singlet

by dye-sensitized

and result

been

by the NADPH dependent

was refuted

shown

acid

and convincingly

The ceric been

generated

has been

in biological

consequence.

presented

('02)

singlet was presented

the decomposition an emission

a OCl--H202

spectra

aqueous

DPIBF, Diphenylisobenzofuran; chromatography; DABCO,

354 ISSh’

0006-291X

BIOCHEMICAL

Vol. 76, No. 2, 1977

system

known

to singlet It

to produce oxygen

shown

unambiguous

Materials

that

evidence

conditions

Nonetheless

linoleic

by heme and hemoprotein

due to the production

under

oxygen.

RESEARCH COMMUNICATIONS

they

assigned

the emission

(9).

was previously

decomposed

singlet

AND BIOPHYSICAL

of radical

to a range oxidizing

is presented

similar

to that

acid

hydroperoxide of products

species

for

singlet

which

could

oxygen occur

(10).

was rapidly complex

In the

formation

in nature following,

by this

reaction

physiologically.

and Methods

The substances used in these studies were obtained from the following sources: 2,5-diphenylfuran, Eastman Organic Chemicals; trans 1,2-dibenzoylethylene, 1,3-diphenylisobenzofuran and 0-dibenzoylbenzene, Aldrich Chemical Company. The substances were dissolved in acetone. Ceric ammonium sulfate was obtained from Sigma Chemical Company and was dissolved in 1 N HCl. Methemoglobin was obtained from Sigma. Linoleic acid hydroperoxide (LAHPO) was prepared by the method of O'Brien Formation of LAHPO, 1 mM type oxygen uptake from FM LAHPO and

O2 Oxygen evolution from a reaction mixture containing 1 mM ceric ammonium sulfate and 0.01 N HCl was monitored with a Clark electrode at 20°C in a reaction vessel of 1.9 ml capacity. Oxygen a reaction mixture containing 50 mM Tris HCl buffer (pH 7.5), 100 1 M methemoglobin was monitored in a similar manner. r Chemiluminescence of the above reaction systems (5 ml) was monitored in the dark by photomultiplication methods using a Beckman Scintillation Counter (Model LS233) operated in the out-of-coincidence mode (11). Counting vials were stored in the dark before use. The reaction was initiated by adding hydroperoxide. Scintillation counts were recorded every 10 seconds for the first minute and every thirty seconds thereafter. Systems containing D20 were adjusted to a pH of 0.3 higher than H20 containing systems so as to give an effective pD the same as the pH of the H2 0 containing systems.

Oxidation of 1,3-diphenylisobenzofuran (DPIBF) DPIBF oxidation was measured spectrophotometrically by recording the decrease in absorbance at 420 nm of the reaction system (3 ml) containing 100 M DPIBF after initiation of the P Negligible reaction by the addition of hydroperoxide. oxidation occurred in the absence of hydroperoxide or methemoglobin. Oxidation of diphenylfuran (DPF) Oxidation of DPF in a reaction system (3 ml) containing 3.3 M DPF and 50 M LAHPO was measured spectrofluorimetrically by r at recording the dtcrease in emission at 368 nm (slit 6 my) with excitation 333 nm (slit 6 mp> using a Perkin Elmer fluorescence spectrophotometer MPF 2A. The reaction was started with 0.5 p methemoglobin. Negligible oxidation occurred in the absence of hydroperoxide or methemoglobin. Thin layer chromatographic analysis svstems were extracted with 1 to 2 the end of each incubation period. for approximately 15 seconds, then the chloroform layer was transferred The chloroform was then removed by residue was transferred totally to

of diphenylfuran and products The aqueous ml of chloroform/ml of reaction svstem at The two phase system was vigorously mixed centrifuged for 10 minutes at 2,000 g and as completely as possible to clean tubes. a flow of N2 gas. The chloroform-soluble thin layer plates (Silica Gel G) with small

355

(10)

BIOCHEMICAL

Vol. 76, No. 2, 1977

TABLE I:

The self

The reaction as described

reaction

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of linoleate

medium contained in Methods.

peroxy

linoleic

acid

radicals

hydroperoxide

and ceric

02 released

chloride

Chemiluminescence

(n moles)

c.p.m. x 10 at 20 sec.

Control

147

460

+ 0.1 mM histidine

141

46

+ 0.1 mM methionine

133

230

+ 0.1 mM nordihydroguaiaretic acid

65

313

+ 0.2 mM nordihydroguaiaretic acid

15

61

130

990

D20 medium

volumes of chloroform. The plates were developed in the solvent system consisting of heptane-dioxane (3/l), air under UV light or after exposure to iodine vapour.

-3

dark for one hour in a dried, and then examined

Thin layer chromatographic analysis of diphenylisobenzofuran and products Prior to use, diphenylisobenzofuran (DPIBF) was purified by TLC on Silica Gel plates using benzene as the solvent. The purified DPIBF was added to the reaction mixture. The chloroform-soluble residue was extracted from the aqueous system and run on TLC as described above using benzene as the solvent. RESULTS The properties acid 1

of the chemiluminescence

hydroperoxide

02 is responsible

to be singlet medium in which oxidant

by eerie

ions

is

was the marked

oxygen

traps

'02 has been

nordihydroguiaretic

(12)

shown

in Table

inhibition

to have

also

I.

Evidence

enhancement

a longer

markedly

356

the oxidation

by histidine

and the marked

shown acid

accompanying

lifetime

inhibited

of linoleic suggesting

and methionine

that known

in a deuterated (13).

The anti-

the chemiluminescence

BIOCHEMICAL

Vol. 76, No. 2, 1977

Singlet

TABLE II:

acid

oxygen

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

formation

hydroperoxide

during

methemoglobin

catalyzed

linoleic

decomposition.

The reaction medium contained 0.1 mM linoleic acid hydroperoxide and 1rM methemoglobin in 50 mM Tris HC1 buffer (pH 7.5) for oxygen uptake and chemiluminescence measurements. DPIBF oxidation was followed at 420 nm with 100 rM DPIBF in the medium. The medium for DPF oxidation contained 3.3 pM DPF, 50 FM LAHPO and 0.5 TM methemoglobin. Methods are described in the experimental section.

at 20 sec.

DPF nmoles oxidized in 20 sec.

Chemiluminescence c.p.m. x 10 -3

nmolesjmin. 02 uptake

DPIBF nmoles/min

Control

137

736

4.6

120

+ 20 mM DABCO

134

1615

2.2

110

+ 1 mM azide

109

149

1.7

111

70

227

2.7

95

100

290

62

35

0.4

11

64

61

0.2

13

+ 10 mM histidine + 50

M bilirubin r + 50 rM nordihydroguaiaretic acid

+ 50 rM butylated hydroxyanisole + 100

M DPIBF r + 100 rM DPF

423 108

605

D20 medium

148

1402

7.1

95

17

23

0.2

6

no hydroperoxide

as would for

the

be expected singlet

if

oxygen

was released

by reactions

was followed

by a slower

self

reaction

formation.

Oxygen

of the peroxy oxygen

of the peroxy

uptake.

electrode radicals From Table

357

radicals studies

during I it

were showed

the

first

responsible that minute

can be seen

that

oxygen but

BIOCHEMICAL

Vol. 76, No. 2, 1977

histidine

and methionine

oxygen

released

formation. that

however

indicating

the lifetime

oxygen

of peroxy

Chemiluminescence hydroperoxide moreover

or cuprous for

during ions

the

(DABCO),

medium

(15).

diphenylfuran

(6)

Chemiluminescence markedly

inhibited

involved

in

the

radicals

and histidine

(12)

cannot

or tert-butyl

hydroperoxide

evidence

that

chemiluminescence

linoleic

to form

during

inhibited

dimol

the

medium.

Antioxidants

peroxy

and Ingold

radicals (8),

of this

were

tertiary no chemi-

and methemoglobin.

can be attributed

the

-2.2

of cumene hydroperoxide

by hematin

during

responsible

chemiluminescence.

In confirmation

the chemiluminescence

cobaltous

in an aqueous 1O2 traps

and the

that

to Howard

catalyzed

emission

(17)

suggesting

02,

1 02 is

that

and

No chemiluminescence

by manganous,

in a deuterated

1

II)

by 1,4 diazobicyclo[Z

the decomposition

was observed

acid

oxidation

dibenzoethylene,

rate

of oxidation

A stoichiometry

a

1

observed

This

to '02.

is

No

decomposition

of H202 catalyzed

in a reaction

mixture

methemoglobin 02 product

of diphenylfuran of 10.2

of diphenylfuran.

containing

products

hydroperoxide,

cis

a '02

also

acid

(Table

ions.

enhanced.

by

or methemoglobin.

The only

mole

bilirubin

According

react

ceric

enhances

(16),

enhanced

formation.

of linoleic

suggesting

which

was not

at pH 7.5

stimulation

chemiluminescence

was observed

hematin

(14) azide

luminescence

further

Evidence

or their

medium indicating

of formation

of LAHPO catalyzed

quenchers

'02

with

of

radicals

in a deuterated

decomposition

observed

decomposition

the amount

the peroxy

rate

the

was the marked

the

affect

or methemoglobin

that

was markedly

appreciably

was less

accompanied

1 a 0 2 quencher

The '02

not

or their

or by glutathione.

octane

did

release

than

the chemiluminescence

peroxy

they

by hematin

was much greater

not affect

radicals

also

catalyzed

was observed

did

that

Furthermore

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

moles

However

From Table

was inhibited

The only

as shown

reaction

were

product (DPIBF)

in Table

358

II,

II

it

corresponded

required

observed

corresponded the

oxidation

to

can be seen that

by DABCO, histidine

of hydroperoxide

1,3 diphenylisobenzofuran

product.

(6).

and diphenylfuran

containing

the

and azide

to oxidize in a reaction

one mixture

to dibenzoylbenzene, of DPIBF when

assayed

BIOCHEMICAL

Vol. 76, No. 2, 1977

at 420 nm was not was slower indicate

in

affected

the

results

uptake

was inhibited

luminescence

quenchers

acid

it

chemiluminescence

(19)

uptake

for

__,

yield

is

likely

appreciably

their

by the

effects

on the rate

by DPIBF confirms

(8) by the

the

concerted

1

that

acid

on chemi-

of decomposition Furthermore

that

following

self

the

DPIBF undergoes

mechanism,

reaction

-----+

of secondary

R(H)OH + RO + '02. by this

reaction

of diphenylfuran

hydroperoxide

originally

,

O2 production

of 10.2% was observed

of the oxygen

hydroperoxide

by ceric

pathway

release ions

during

however

catalyzed during

that

peroxy In the

explains

the

accompanies

by hemoproteins

the decomposition

ZR(H)OO'

The formation

of peroxy

radicals

hydroperoxide

catalyzed

by methemoglobin

radicals

The formation also

explain

with

oxygen

(21):

Alternatively

radicals

observed

and enter

__3

the alkyl

rapid

during

by the oxygen

autoxidative radicals

decomposition

the or ceric

of hydroperoxide

of linoleic

R(H)OOOOR(H)

probably

ZR(H)O'

of linoleic

involves

+ Oz. acid

the reaction

of

ROO' + ROH. scission uptake chain

could

acid

non-concerted

------+

the decomposition

RO' + ROOH -----+

of alkyl the

the

may be due to the following

(20):

(22).

affected

oxygen

by methemoglobin.

Most

alkoxy

catalyzed

can be seen that

by methemoglobin.

R(~)0000R(H)

of linoleic

A '02

it

that

to effects

and the cooxygenation

decomposition

radical

of DPIBF to

the methemoglobin

II

and indicates

may be formed

ZR(H)OO'

catalyzed

by antioxidants

system.

oxygen

study

ions

but was not

catalyzed

of oxygen

by Russell

salts.

the reaction

the autoxidation

From Table

or traps

hydroperoxide

in this

Singlet

uptake.

ceric

not be attributed

enhancement

autoxidation

with

by antioxidants

could

of linoleic

present

Furthermore

inhibition

may catalyze

reaction

in oxygen

oxygen

radicals:

The ready

radicals

to the

reaction

proposed

or methionine.

(18).

In contrast

marked

medium.

peroxy

dibenzoylbenzene

singlet

by histidine

a deuterated

that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of these as the alkyl

sequence

be formed

359

again

alkoxy

radicals

radicals as peroxy

by the abstraction

react

would rapidly

radicals of a

BIOCHEMICAL

Vol. 76, No. 2, 1977

hydrogen

atom from

dihydroperoxide

the hydroperoxide

Thus

of '02

regarding

Smith

normally

formed

radicals

react the

from

(7)

1

using

by free with

02 is

faster

1 of 02 products

lack

which

tetraphenylcyclopentadienone studies,

the

initiated

'02

trap,

enhanced

by a deuterated

Care must

therefore

be exercised

The rapid

considerable been

and lead

Acknowledgement:

during

to

some of the

microsomal

lipid

as a 102 trap

found

autoxidation.

Presumably

than

was used

as the

buffer in the

relevance.

The oxygenation

of '02

The enzymic

explanation

may

(4)

in

underwent

or inhibited

by '02 traps

radicals

at a neutral

the peroxy

trap.

(18).

choice

products

reaction

1,3-diphenylisobenzofuran radicals

peroxidation.

only

does l0 2. A similar in a lipoxygenase

by hemoproteins

physiological

may explain

of 1 O2 and peroxy

formation

catalyzed

demonstrated

Canada

radicals

obtained

by the peroxy

therefore

decompcsition

formed

catalyzed

cholesterol

for

formation.

alkoxy

radicals

cholesterol

radical

exist

autoxidation

the peroxy

whether

and Teng

In our

by the

RESEARCH COMMUNICATIONS

formation.

The formation controversy

AND BIOPHYSICAL

quenchers

or traps.

to demonstrate

l0

during

pH should formation

was not

lipid prove

2

peroxide to be of

of 102 has recently

(23). This

and the National

work

was supported

Research

by the National

Cancer

Institute

of

Council.

References 1. Foote, C.S. (1976) in "Free Radicals in Biology", (ed. W.A. Pryor), Vo 1. 2 Academic Press, New York. 2. Weishaupt, K.R., Gomer, C.J. and Dougherty, T.J. (1976) Cancer Res. 36, 2326-2329 3. Chan, H.W.S. (1971) J. Amer. Chem. Sot. 93, 2357-2358 4. Baldwin, J.E., Swallow, J.C. and Chan, H.W.S. (1971) J. Chem. Sot. Chem. Commun. 1407-1410 5. Pederson, T.C. and Aust, S.D. (1972) Biochem. Biophys. Res. Commun. g, 789-795 6. King, M.M., Lai, E.K. and McCay, P.B. (1976) .I. Biol. Chem. 250, 6496-6502 7. Smith, L.L. and Teng, J.I. (1974) J. Amer. Chem. Sot. 96, 2640-2641 8. Howard, J.A. and Ingold, K.U. (1968) J. Amer. Chem. Sot. 90, 1056-1058 9. Nakano, M., Takayama, K., Shimizu, Y., Tsuji, Y., Inaba, H. and Migita, T. (1976) J. Amer. Chem. Sot. 98, 1974-1975 10. O'Brien, P.J. (1969) Can. J. Biochem. 5, 485-492 11. Allen, R.C., Stjernholm, R.L. and Steele, R.H. (1972) Biochem. Biophys. Res. Commun. 47, 679-684 12. Nilsson, R., Merkel, P.B. and Kearns, D.R. (1972) Photochem. Photobiol. 16, 117-124

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Vol. 76, No. 2, 1977

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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