Synthesis of spin labelled 2,4-dinitrophenols and their activities in the uncoupling of oxidative phosphorylation in rat liver mitochondria

BIOCHEMICAL

Vol. 48, No. 5, 1972

Synthesis and Their

of Spin Labelled

Activities

Hsia,

W.L.

2,4-dinitrophenols

in the Uncoupling

Phosphorylation J.C.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in Rat Liver

Chen, L.T.

Department

of Oxidative

Mitochondria

FTong, R.A.

Long and W. Kalow

of Pharmacology,

Faculty

of Medicine,

University

of Toronto,

Toronto

181, Ontario, Canada.

Received

July

25,

1972

Summary Two spin labelled dinitrophenols [l-Hydroxyl-5-N(l-oxyl-2,2,5,5-tetramethyl-3-amino-pyrrolidinyl)-2,4dinitrobenzene, and 1-Hydroxyl-5-N-(1-oxyl-2,2,5,5tetramethyl-3-amino-methylpyrrolidinyl)-2,4-dinitrobenzene] were prepared and found to retain full activity in the uncoupling of oxidative phosphorylation in rat liver mitochondria. Spin labelled 2,4-dinitrobenzene analogues are inactive. Several

hypotheses

the uncoupling chondria

of

oxidative

by various

A clear

difficult

the uncoupler

a detailed

ation

concerning

Copyright All rights

the behavior

@ 1972 by Academic Press, reproduction in any form

of

labelled

lipids

the organization

model and biological that

knowledge

in the mitochondrial

The use of spin

Inc. reserved.

vary

phosphorylation among these

membranes 5-8 . of an uncoupler

1273

for

in mito-

l-4 , and they

oxidative

distinction

without

to account

phosphorylation

uncouplers

to the mechanisms of favor.

have been proposed

according the

authors

hypotheses of the

is

behavior

of

membrane. has provided and order Thus,

of it

is

inform-

lipids

in

very

likely

in model and mitochondrial

Vol. 48, No. 5, 1972

BIOCHEMICAL

membranes could label

to the

be analyzed

However, does not

the

spin

labelling

ivity

of

the

uncoupler.

labelled

approach

to

study

of

oxidative

phosphorylation

chondrial

membranes 9 .

preparation

present of

it

is

would

important

to show

alter

the

preparation

the mechanism of uncoupling as the structure

communication labelled

a spin

actof

such

open up a new experimental

as well

two spin

conjugating

seriously

The successful

uncoupler

the

In the

by covalently

uncoupler.

that

a spin

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

we report

the

2,4-dinitrophenols

of of mito-

successful (DNP)

Ho&N*2Ho&-No NH

NH /

& r

0

DNP-SL(S)

which

retain

phosphorylation

DN P-methylene-SL(5)

full

activity in rat

in the uncoupling liver

of oxidative

mitochondria.

MATERIALS AND METHODS Synthesis

of of

1-hydroxyl-5-N-(1-oxyl-2,2,5,5-tetra-

methyl-3-amino-pyrrolidinyl)-2,4-dinitrobenzene A mixture

of

50 mg of

1-fluoro-5-N-(1-oxyl-2,2,5,5-tetra-

1274

(DNP-SL(5)):

BIOCHEMICAL

Vol. 48, No. 5, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

10

methyl-3-aminopyrrolidinyl)-2,4-dinitrobenzene of 10% caustic the

reaction

acid

soda was refluxed mixture

was acidified

and the precipitate

extracted

with

ethyl

acetate

The total

was 32 mg

yield),

methanol.

Molecular

Mass spectroscopic Synthesis

2 hours.

with

After

diluted

was filtered.

was obtained. (64%

for

The filtrate

m.p. weight

analysis

of the

crop

of product

red crystalline

solid

recrystallization

calculated

for

from

C14H19N406 = 339.33.

= 339.

of 1-hydroxyl-5-N-(l-oxyl-2,2,5,5-tetramethyl-

3-amino-methylpyrrolidinyl)-2,4-dinitrobenzene SL(5)):

was

183O after

found

cooling,

hydrochloric

and an additional

yield

and 10 ml

(DNP-methylene-

The compound was similarly

prepared

from l-fluoro-5-N-

(1-oxyl-2,2,5,5-tetramethyl-3-aminomethylpyrrolidinyl)-2,410 and the yellow

dinitrobenzene (85% yield)

has m.p.

methanol-water. H, 5.95;

Analysis

N, 15.85.

The pK values spectrophotometric and found (5)

146-148"

solid

obtained

recrystallization

calculated

for

from

C15H21N406 = C, 51;

H, 6.06;

N, 16.03.

were determined

by a modification

method described

by Rosenburg

to be 4.8 for

labelled

after

Found = C, 51.11;

as compared to 4.1 Spin

crystalline

DNP-SL(5) for

and 5.6 for

of

the

and Bhowmik 11

DNP-methylene-SL

DNP.

2,4-dinitrobenzene

(DNB) analogues

[N-(l-oxyl

2,2,5,5-tetramethyl-3-aminopyrrolidinyl)-2,4-dinitrobenzene

and

N-(l-oxyl-2,2,5,5-tetramethyl-3-aminomethyl-pyrrolidinyl)-2,4dinitrobenzene]

were synthesized

Preparation isolated

of mitochondria:

according

modification. 0.07 M sucrose,

by Hsia 10 .

as described Rat liver

mitochondria

to the method of Hagihara 12 with

The preparation

medium contained

1.0 mM tris-hydroxymethylamino

1275

was

slight

0.21 M mannitol, methane,

0.1 mM

BIOCHEMICAL

Vol. 48, No. 5, 1972

EDTA, and 0.5% bovine adjusted

serum albumin:

to pH 7.2 with

Oxygen uptake

uptake

monitor

Yellow

Springs,

was carried

Ohio)

out

for 13

by Estabrook

(Yellow

Springs

solution

was

measuring

O2

.

A YSI Model

Instrument

was used in these

Co.,

studies.

in a medium containing

WW4 r 50 mM KCl, buffer,

The method

described system

and the

0.1 N NaOH.

studies:

was that

oxygen

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Inc.,

The reaction

0.15 M sucrose,

50 mM tris-hydroxymethylamino

and 10 mM phosphate

53

5 mM

methane-HCl

buffer. RESULTS

Uncoupling ative

oxygen-uptake

mitochondria of

activity

1.8

After

spin

experiment

is

succinate addition

DNP-SL(5)

respiration

and further

addition

respiratory

rate uncoupled

Table labelled ative from

that

6.4 to

8.0.

coupled

The spin

DNB analogues

at

are com-

activities

of oxid-

possess

as DNP through-

we have used spin concentrations

and found

activity.

DISCUSSION The observation

that

the uncoupling

1276

the

at pH ranging

DNP molecules

comparable

possess no uncoupling

of

increase

uncoupling

mitochondria

labelled

6.

of DNP and spin

maximal

liver

of

DNP.

As a control

studied.

index

a stimulation

concentrations

to achieve

The

a P/O ratio

the mitochondria

labelled

in rat

with

control is

1.

of ADP does not

the same uncoupling

the pH range

they

spin

1 compares the

phosphorylation

labelled

shown in Figure

there

that

by the

DNP required

approximately out

indicating

A represent-

DNP:

and a respiratory of

pletely

labelled

are shown to be tightly

for the

of

activity

is

ex-

BIOCHEMICAL

Vol. 48, No. 5, 1972

Table

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Uncoupling activities Optimal concentrations coupling of oxidative

1.

DNP

PH

of DNP and spin labelled DNP. required for maximal unphosphorylation.

DNP-SL(5) (concentrations

DNP-Methylene-SL(5) in

PM)

6.4

14

18

21

7.4

41

45

51

8.0

95

106

122

mito J

Figure

ADP

1. The effect of spin labelled DNP on mitochondrial respiratory control. The oxygen-uptake reaction was carried out in 3.0 ml of the reaction medium as described under "Materials and Methods". 0.2 ml of mitochondria (15 mg of protein per ml), 0.03 ml of 1.0 M succinate, 0.1 ml of 5.0 mM ADP, 0.25 ml of 640 uM DNP-SL(5), and 0.1 ml of 5.0 n@l ADP were added to the reaction mixture as indicated in the figure. The reaction medium contains 240 mumoles O2 per ml.

elusively phenols

associated but

demonstrates

not with that,

with the

the spin

labelled

2,4-dinitrobenzene

(a) the phenolic

1277

2,4-dinitroderivatives

group of DNP is

essential

Vol. 48, No. 5, 1972

for

BIOCHEMICAL

the uncoupling

activity,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and (b) the

spin

labelling

does not

uncouple

oxidative

the

labelled

DNP and DNB compounds were found

spin

readily

reduced

by the mitochondria

strate-linked.

This

action

between

of the

respiratory

account

of

phosphorylation.

the

label

chain

these

under

experiments

ideal

motional

ship

after

of

spin the spin

membranes in an attempt

between

the inner

conservation'.

mental

approach

drug

full

we have studied

energy

of

will

conditions

characteristics

chondrial

and the

action

could

is

and other

sub-

inter-

A detailed elsewhere. work is

of the present

biological

of

activity

a com-

labelling. orientation, labelled

distr

bution

the

relation-

membrane structure that

to the

biologically

and

DNP in model and mito-

to understand

possible

be applied

is

component(s)

be reported

mitochondrial It

both

to be

direct

particular

implication

pound can be preserved Recently

due to the

in the mitochondria.

The most significant that

However,

and the reduction

is presumably

spin

per -se

a similar

study

active

of

and experi-

the mechanism

substances.

ACKNOWLEDGEMENT This

research

MRC-DA6 from National

was supported

the Medical

Health

Research

and Welfare

in part Council

by Grant

MA-4129

and Department

and of

of Canada. REFERENCES

1.

2. 3. 4. 5. 6. 7.

Mitchell, P. Chemiosmotic Coupling and Energy Transduction, Glynn Res. Ltd., Bodmin, Cornwall (1968). Van Dam, K., E.C. Slater, Proc. Nat. Acad. Sci. U.S.A., 58, 2015 (1967). Kinbach, E.C., J.Garbus, Nature, 221, 1016 (1969). Green, D.E. and H. Baum, Energy andhe Mitochondrion, Academic Press, New York (1970). and I.C.P. Smith, Biochim. Hsia, J.C., H. Schneider Biophys. Acta, 202, 399 (1970). Seelig, J., J. Amer. Chem. Sot., 92, 3881 (1970). and H.M. McConnell, J. Amer. Chem. Sot., Hubbel1,W.L. 93, 314 (1971).

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Vol. 48, No. 5, 1972

8. 9. 10. 11. 12. 13.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Jost, P., L.J. Libertini, V.C. Herbert and O.H. Griffith, J. Mol. Biol., 2, 77 (1971). Hsia, J.C., W.L. Chen, R.A. Long, L.T. Wong and W. Kalow, manuscript in preparation. Hsia, J.C., Ph.D. Thesis, University of Hawaii (1968). Rosenberg, B. and B.B. Bhowmik, Chem. Phys. Lipids 3, 109 (1969). Hagihara, B.; Biochim. Biophys. Acta, 46, 134 (1961). Estabrook, R.W., Methods in Enzymology, 10, 41 (1967).

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