Fluorescence studies of a cytochrome c mixed phospholipid complex

Vol. 51, No. 1,1973

BIOCHEMICAL

FLUORESCENCE

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

STUDIES

OF

MIXED PHOSPHOLIPID Laurence

A

CYTOCHROME

COMPLEX+

S. Kaminsky,

Jean

J.

Kathryn

M. Ivanetich

Henderson

Department of Physiology and Medical University of Cape Town Medical Cape Town, South Africa Received

January

C

and

Biochemistry, School,

16,1973

Summary : The fluorescent probe 8-anilino-1-naphthalene sulphonic acid (ANS) has been used to demonstrate the accessibility of the phospholipid complexes heme of cytochrome c - mixed mitochondrial to the solvent. Contrary to earlier reports fluorescence techniques using ANS do not detect redox induced conformational changes of these complexes. Cytochrome lipids

2 in

of the

phospholipid

mitochondrion

changes

oxidation

state

(5),

occurrence

cytochrome reported

that

increased

acid

intensity

from

these

University

Copyright 0 19 73 by Academic Press. Inc. All rights of reproduction in any form reserved.

of the fluorescence

phospholipid

the

in model redox

of for

South

African

of Cape Town Staff

40

of

investigated

changes.

8-anilino-l-

hydrophobic

c - phospholipid

reduction

mitochondrial

by grants and the

undergoing

a probe

changes

recently

complexes

(ANS),

confor-

following

effects

2 for

extensive

conformational

by cytochrome following

of the

(6) have

phospho-

as a model

c undergoes

et al.

the

cytochrome

investigated

fluorescent

and extended

c - mixed

+Supported Council

the

sulphonic

reinvestigated chrome

similar

was enhanced

further

Azzi

with

Consequently

In view

cytochrome

2 - phospholipid

naphthalene ments,

of

been

c (3,4).

that

complexed

(1,2). have

cytochrome

mational

They

is probably

complexes

endogenous

the

vivo

environ-

complexes

complexes. studies

We have on cyto-

complexes

Medical

and

using

Research

Research

Fund

the

VoL51,No.

heme induced We have

quenching

the

following:

by cytochrome

protein

is

in

The fluorescent

the

of the

heme iron.

changes

reported

identical,

form

or complexed

is not

Previous

complexes

reducing

properties

presence

of cytochrome

are,

purchased with

Miles

potassium

purified heart chrome

mitochondrial

and their

16 lysine

Fluorescence

Water spectra

spectra

were

complex

and complexed

free

and the

or solubilized

Merck,

in

as its

bacteriology

residues

phospho-

sodium

magnesium grade.

salt.

Dicarboxy-

cytochrome

(9) were prepared

was distilled

c

as described

and deionized.

were determined

with wavelength

41

cyto-

Both

twice

over

beef

(Ivanetich,

2 (8) and succinylated

scanned

with

and

described

The complex

The excitation

spectrofluorometer.

was oxidised

39.4% phosphatidylethano-

dispersed

modified

England,

Mixed

ANS was recrystallised

literature.

of the

Fe)

G-25 column.

9.5% cardiolipin,

was from

0.425%

a Sephadex

submitted).

cytochrome

emission

95% pure,

as previously

methylmethionine

the

I,

dithionite

were ultrasonically

in

independent

sodium

lipids

at over

-c

of the

with

and 51.1% phosphatidylcholine.

deoxycholate

a function

or reduced

lamine

Sodium

of cytochrome

and are almost

phospholipids

comprised

deoxycholate.

not

Maidenhead,

through

and Kaminsky,

phospholipid

reduction

and Methods

Seravac,

c were prepared

Henderson

change

(6) on ANS fluorescence

2 (Grade

ferricyanide

by passage

valency

-c

2.

cytochrome

from

of cytochrome

following

in fact,

of ascorbate

of whether

to phospholipids.

presence

altered

Materials heart

the

results

of ANS

irrespective

to be due to ascorbate

- phospholipid

Horse

The quenching

of ANS in

complexes

(7).

2 is

free

intensity

- phospholipid

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of ANS fluorescence

demonstrated

fluorescence the

BIOCHEMICAL

1,1973

the

range

a Perkin

Elmer

203

was 340 nm and 440-520

run (6).

BIOCHEMICAL

Vol. 51, No. 1,1973

All

assay

0.02

mixtures

contained

M Tris-HCl

buffer,

deoxycholate, indicated

0.275

pH 7.4

cytochrome in the

c,

figure

both

enhance

470 run.

A solution

enhances

the

dispersed

2,

deoxycholate

ANS fluorescence

which

the

complex of the which

provides

of the from the

enhanced

1).

complex

or cytochrome

solutions to

it

is

the

an identical

from

is clear the

lipid

from

these

cytochrome

(5) or to some part

of the

surface

of the

would

not

it

occurs

if

to the

prevent via

heme.

electron the

exposed

the

of

results

2 in the

to

electrons

quench

of hemoproteins

solvent

for

(Figure

dispersions

of the

access

extent,

complex

heme moeity

around

Addition

of ANS in

due to the

arising

does either

(Figure

access

of ascorbate

to mixed

of ANS and cytochrome the

effectiveness with

than

the

Thus

exposed

edge

protein the

transfer

presence

to and

heme edge,

or from

surface.

Addition solutions

alone

(7) it

prevent

heme iron,

protein

extent

2 - phospholipid

sheath

phospholipids

the

however,

quenching

to that

Since

phospholipid

heme

in deoxycholate,

buffered

slight

fluorescence

does not

band of ANS at

c - phospholipid

cytochrome

alone.

as

and deoxycholate

ANS fluorescence

relative

quenches

that

the

the of

M sodium

intensity

to a lesser

to aqueous

Dispersions

phospholipid

phospholipids

fluorescence

cytochrome

for

of 0.025

and complex

or deoxycholate

quenches

when corrected 1).

mixed

of phospholipid

phospholipid

M sucrose,

and Discussion

the

phospholipid

c plus

and combinations

ANS fluorescence

of cytochrome

0.025

legends.

dispersed

markedly

M mannitol,

phospholipids

Results Ultrasonically

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

increasing

fluorescence of ascorbate quantities

phospholipids c as described

emission in

dispersed

of the

enhancing

of phospholipid 42

by Azzi

in et al.

ANS at 470 run.

ANS fluorescence until

a plateau

(6)

The increased was

VoL51,No.

BIOCHEMICAL

1,1973

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

,

0

I 460

LLO

I Leo WAVELENGTH

1 500 (nm)

(mg Plml)

Fig. 1.

Fig. 2. Figure 1. Fluorescence emission spectra of ANS in solutions of: 0.025 M sodium deoxycholate; (-• -9 0.03 mg P/ml (0.75 mg (- --) phospholipid/ml) mixed mitochondrial phospholipids in 0.025 M 1 13 uM ferricytochrome c in 0.025 M sodium deoxycholate; ((** ** 1 cytochrome 2 - phospholTpid complex sodium deoxycholate; 113 ~.IMcytochrome c and 0.03 mg P/ml phospholipids) in 0.025 M sodium deoxycholate. 7.2 x 10e5mM ANS, 50 mM ascorbate. l

Figure 2. The influence of mixed phospholipid concentration on the enhancing effect of ascorbate on ANS fluorescence in the presence of: ( 0 1 13 uM cytochrome c and ultrasonically ( U 1 ultrasonically dispersed phosphodispersed phospholipid; lipid alone. 7.2 x 10B5mM ANS, 50 mM ascorbate.

reached

(Figure

ascorbate

is not

carbohydrates responses, lipid Attempts

2).

We have

due to its

to provide

apparently complete

also

behaved

heme is identically

the

of

cytochrome

significantly with

altered cytochrome

43

of

fluorescence.

2 in

similar

the

the

ANS.

ANS with

the

environment

cytochrome system.

using

of the

in which

this

of

c by ascorbate

reduction

from

elicit

for

in

2,

other

properties

cytochrome

resulted

of

since

environment

the

effect

acid

micellar

disappearance

Dicarboxymethylmethionine of the

properties

a more hydrophobic reduction

this

and glucuronic

by altering

to mimic

a consequent

reducing

such as glucose perhaps

dithionite

now shown that

c (lO,ll), Consequent-

Vol. 51, No. 1,1973

ly

structural

are not

features

likely

to play

fluorescence 2) is

intensity

not

complex mixed

of reducing

complexes

irrespective

of

the

reports

with agent

It

is

enhance oxidation

effects

mixture

the

results

The slightly

from

it

when cytochrome

detected

using

for

presence

use of this

for

the

result

ANS interfrom

the

effectiveness

of ascorbate

the

of cytochrome

and that

to detect

cannot the

known be

previous

and the

transport

relevance cannot

substantiated. References 1. D.E. Green (1963).

and S. Fleischer,

Biochim.Biophys.Acta,

44

report

such conformational

complexes

of electron

of the

changes

changes

in

2

properties

structural

redox

technique

mechanism

the

altered

protein.

2 - phospholipid

c and that

significantly

of the

technique

same extent

completely

micellar

c undergoes

c -

phospholipid

the

that

fluorescent

cytochrome

of such changes

in

to the

omitted

presence

alterations

to occur

the

the

is apparent

this

2 is

from

cytochrome

cytochrome

(6) are only

on the

c,

fluorescenue

cytochrome

changes

are not

in

due to the

In conclusion

changes

with

submitted),

and the

that

of the

diminished

ANS fluorescence

concerning

complex

cytochrome

in the clear

of ascorbate

reaction

phospholipids

thus

induced

when cytochrome

results

reduced

were removed

state

Finally

possibly

c-phospholipid

does not

ANS fluorescence

action.

enhancing

(Figure

and Kaminsky,

no difference

of redox

of non-specific

2).

c , which

Henderson

constituted

was

phospholipid

(Figure

concentration

of a cytochrome

cytochrome

c

The enhanced

lipid

formation

was observable.

previous

effects.

increasing

to autoxidise,

emission

these

of cytochrome

results.

traces

c permitted

in

(Ivanetich,

system

all

a role

succinylated

identical

When the

heme environment

on the

phospholipids

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the

with

dependent since

yields

which

BIOCHEMICAL

70,

554

be

VoL51,No.

1, 1973

BIOCHEMICAL

AND BIOPHYSICAI

RESEARCH COMMUNICATIONS

2. K.S. Ambe and F.L. Crane, Science, 129, 98 (1959). 3. M. Reich and W.W. Wainio, J.Biol.Chem., 236, 3058 (1961). 4. H-K. Kimelberg, C-P. Lee, A. Claude and E. Mrena, J-Membrane Biol., 2, 235 (1970). 5. T. Takano, R. Swanson, O.B. Kallai and R.E. Dickerson, Cold Spring Harbour Symposium on Quantitative Biology, 36, 397 (1971). 6. A. Azzi, S. Fleischer and B. Chance, Biochem.Biophys.Res.Commun., 36, 322 (1969). C-G. Rosen, FEBS Lett., 5, 158 (1970). 2 A. Schejter and I. Aviram, J.Biol.Chem., 245, 1552 (1970). 9. K. Wada and K. Okunuki, J.Biochem., 64, 667 (1968). 10. L.S. Kaminsky, V. Miller and K. Ivanzich, Biochem.Biophys. Res.Commun., 48, 1609 (1972). 11. R.M. Keller, I. Aviram, A. Schejter and K. Wfithrich, FEBS Lett., 20, 90 (1972).

45