Vol. 84, No. 1, 1978 September

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

14, 1978

Pages

THE EFFECT OF PHOSPHOLIPID

VESICLES

ON THE KINETICS

254-260

OF REDUCTION OF

CYTOCHROME C John B. Cannon Department

of Chemistry, DeKalb,

Received

July

and James E. Erman Northern

Illinois

Illinois

University,

60115

17,1978

SUMMARY The rate of reduction of cytochrome c by ascorbate and by 2-amino-4hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine was examined as a function of ionic strength and of binding to phospholipid vesicles (liposomes). Binding of cytochrome c to liposomes, which occurs at low ionic strength, decreases the rate of reduction by ascorbate by a factor of up to 100, which can be primarily explained on electrostatic grounds. In the absence of liposomes, kinetics of reduction by the neutral pteridine derivative showed no ionic strength dependence. Binding of cytochrome c to liposomes increased the rate of reduction by pteridine. An estimation of the binding constant of cytochrome c to liposomes at 0.06 M ionic strength, pH 7, is given. Cytochrome present

between

the membrane binding

c, an important the outer

on its

(liposomes)

in this

field

have

cytochrome

c with

such liposomes

attraction

between

the positively

liposomes.

High

evidence,

however,

partial We report kinetics

ionic that

strength the

of the

herein

an examination

Abbreviations: pteridine; Tris,

heart

system

yet

for

charged disrupts

the the

examination

nature

c molecule effect

c reduction

hydrophobic,

of liposome by the

lipid binding

negatively

DMPH4, 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydroTris(hydroxymethyl)aminomethanc; Pi, phosphate.

0 1978 by Academic Press, Inc. of reproduction in any form reserved.

254

question.

interaction

(2,3).

the

of

The

of this

c and the

into

is

vesicles

electrostatic,

is partly

of the

(1).

of the

complexes

chain,

The effect

understood

cytochrome the

transport

of phospholipid

is predominantly

0006-291x/78/0841-0254$01.00/0 Copyright All rights

not

shown that

cytochrome

cytochrome

electron

membranes.

surface

interaction

penetration

of horse

however,

external

a good model

of the

mitochondrial

is,

c to the

provides

Most workers

and inner

reactivity

of cytochrome

component

of

i.e.,

a charge

negatively There that

is there

bilayer on the charged

charged some is (4-6).

BIOCHEMICAL

Vol. 84, No. 1, 1978

reductant

and also

ascorbate,

strength

dependence

revealed

explained

purely

MATERIALS

AND METHODS

AND BIOPHYSICAL

the neutral behavior

by electrostatic

reductant

for

the

RESEARCH COMMUNICATIONS

DMPH 4'

reduction

A study

of ionic

by DMPH4 that

cannot

be

binding.

Fresh horse heart cytochrome c (Sigma, Type VI) was used without further purification. Solutions of ascorbic acid (Fisher) were neutralized with KOH and the concentration determined by iodometric titration (7). DMPH4 was purchased as the hydrochloride (Aldrich); solutions of DMPH4 were prepared by weight and neutralized with KOH under nitrogen. Phospholipid vesicles were prepared by homogenizing the crude soybean Asolectin (Associated Concentrates) in water, sonicating with a Branson Sonifier for 45 minutes, and centrifuging at 160,000 g for 30 minutes. The slightly turbid supernatant which was used for the experiments could be analyzed by a variation of the Fiske-Subbarow method (81, and generally showed phospholipid content corresponding to 70% of the original weight of asolectin. Kinetics with ascorbate were performed on a Cary 14 spectrophotometer, by observing the increase in absorbance at 417 nm. Kinetics with DMPH4 were performed on a Durrum-Gibson Stopped Flow apparatus with observation at 550 nm. All solutions were deaerated by bubbling with nitrogen immediately before use. RESULTS Figure presence

1A shows the

and absence

ionic

strength

involving chrome

the

phosphate

rate

the

ascorbate (pH 8.01,

previously

Tris-cacodylate

reported:

were

of vesicles

The results

are

Table kinetics never

I gives were

observed,

for

as

a reaction

charged

(pH 7.21, rates

the

in K-Pi/

theory

although

in

is observed

and the positively

systems.

kinetics

by ascorbate

strength,

Debye-HClckel

similar,

of measurement,

biphasic

cyto-

and potassium

in Tris-Cl

were

in good agreement

a summary. monophasic

Under for

in contrast

our

>90% of

to previous

(9,lO). The reduction

vesicles workers (12)

with

c

of ionic

the absence

qualitatively

and methods

reaction;

reports

charged

in any of the buffer constants

conditions

in

consistent

in Tris-Cl

(pH 7) were than

with

increased,

of cytochrome

as a function

in rate

negatively

Results

c.

faster

is

of reduction

of vesicles

A decrease

KN03 (PH 7).

rate

(Figure report

results

of cytochrome 1A)

was

a 15 fold in a negative

c by ascorbate

up to 100 decrease charge

times

(11).

slower Because

on the vesicles

255

in the presence at low the

ionic

composition

at pH 7, the

of phospholipid strength;

previous

of asolectin cytochrome

c-

BIOCHEMICAL

Vol. 84, No. 1, 1978

I

0.0

I 0.2

Ql

AND BIOPHYSICAL

I

I

0.3

0.4

0.0 l/2

I 0.1

RESEARCH COMMUNICATIONS

I

I

0.2

0.3

’ 0.4

25

I

FIGURE 1:

Reduction kinetics of cytochrome c as function of ionic in l-10 mM KPi (pH 7) buffer: KN03 was added to bring ionic strength. A: Cytochrome c (6 PM) + ascorbate. Open Circles: [vesicles] = 0, [ascorbate] = 0.3 m&l Solid Circles: [vesicles] = .7 mq/ml, [ascorbate] = 2 8: Cytochrome c (1.6 nM) + DMPH . Open Circles: [vesicles] = 0, [$MPH4] = 0.04 mM - 1.5 Solid Circles: [vesicles] = 0.18 mq/ml, [DMPH4] = 0.02

TABLE I.

Rate Constants

for

Reduction

of Cytochrome

strength, to required 2 mM. mM - 10 mM. mM. mM - 1 mM.

c by Ascorbate

* k, M-l s-l (this work, without vesicles)

Conditions 0.002 M 0.01 M 0.01 M 0.10 M 0.002 M 0.10 M t

phosphate, pH 7.0 phosphate, pH 7.0 Tris Cl, pH 8.0 Tris Cl, pH 8.0 Tris cacodylate, pH 7.2 Tris cacodylate, pH 7.2

References

given

300

-40-50

49 1300

k, M-l s-l (this work with vesicles) 3.6 32

( 9)

--

450

750 600 --

610 64

10.2 ( 91

160

(10)

3.3 45

in parentheses.

vesicle

complex

charged

ascorbate

As ionic

strength

obtained

in the absence

binding

k, M-l s-l (reported, without vesicles)

would

have a lower

than is

of cytochrome

would increased

rate

of reaction

the more positively to >0.15

of vesicles,

M, the

since

c to phospholipid

high

vesicles

256

with charged

rates ionic (2,3).

the

negatively

"free"

cytochrome

become equal strength

c.

to those

disrupts

At no point

the were

BIOCHEMICAL

Vol. 84, No. 1, 1978

biphasic

kinetics

observed

for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the reduction

of cytochrome

c in the presence

of vesicles. Because

of the

large

c-vesicle-ascorbate nation

might

system,

lB,

absence

of vesicles

lo3 M-1

S-l

over

independent

is

at which

than

cytochrome

in the absence mediate

rate

and unbound

vesicles

from

1B also

strength of vesicles.

c is

to vesicles,

not bound Between

are observed,

0.03

0.02

of ionic

At high the

at pH 7 (l.3)

of 2.8+

0.3 x

in

strength.

In

M) this ionic

rate

constant

is

rate

is

strength

the

(> .l

strength,

inter-

of both

was a biphasic

M),

same as that

due to the reaction

strength

exami-

mM to 1.5 mM, and

M and 0.1 M ionic

apparently

At no ionic

c.

constant

(u = 0.003-0.03

in the absence

for

c by DMPH4 in the

shows the rate

as a function

at low ionic

cytochrome

a rate

of DMPH4 concentration

of vesicles. values

with

area

is uncharged

of cytochrome

Figure

of the cytochrome

a more fruitful

by DMPH4 which

and efficient,

of phospholipid

faster

on the behavior

that

of reduction

strength.

to ascorbate,

5 times

was thought

rate

rapid

a range

effect

of reduction

the

of ionic

the presence contrast

it

be the kinetics

As shown in Figure

is

electrostatic

reaction

bound observed.

DISCUSSION Other

than

specific

ion

vesicle-ascorbate

system

Below

strength,

0.1 M ionic

a slower rates

rate

relative under

amounts these

means that and that

there the rate

than

chrome

c should

0.03

with

of vesicle

is a rapid

range and free that

entirely

The rate

continues

0.03

between

of Debye-HUckel

rate.

Below

in the bound to decrease effects.

257

grounds.

complexes

can form,

of charge

repulsions.

M-0.1

M depend

cytochrome

biphasic

the cytochrome

on electrostatic

c which

kinetics bound

of the cytochrome

reduction

be present

M, because

because

equilibrium

of dissociation

resu 1 ts involving

c-vesicle

strength bound

the

primarily

ascorbate

The fact

the observed

DMPH4 results).

cytochrome

ionic

conditions.

faster

below

in the

(14),

can be understood

of reaction

observed

effects

upon

M ionic

The the

not observed

and free

0.03

have

are present

were

c-vesicle

which

cytochrome complex strength,

c,

is cyto-

form

(as indicated

by the

as ionic

strength

is decreased

Since

ionic

strength

affects

c-

Vol. 84, No. 1, 1978

the binding bound

BIOCHEMICAL

of cytochrome

and free

cytochrome

For the reductant free

cytochrome

ionic

strength,

the

bound

form form

cytochrome

c.

rate

was first

order

tion

rate

rate

The simplicity

This

c from the

strength,

0.06

equation

c

=

is

of free (occupied)

can be obtained, The minimum chrome of total lipid

binding

vesicle

c concentration, binding molecule

sites occupies

and

sites.

concentration

kinetics the

for

were

bound

and the the

the dissocia-

c-vesicle

and free

x 100

system

cytochrome

c

(1)

is

76% free

make possible

of cytochrome

cytochrome

c at

an estimation

c-vesicle

of the

binding,

equation

(2) cytochrome

[c-v]

is

While

accurate

of values

of total is

Between

sites.

of bound

there

of free

a range

s-l).

or as

[VI,

sites,

binding only

that

[c-v1

the concentration

vesicle

s-l)

free

calculations

[cl, (cl,

-1

-1

of 5 mM, at which

DMPH4-cytochrome

obs

-k

constant

KD = where

-k

k bound

(dissociation)

M

1:

bound

These

x lo4

limit

and

of either

k = 5800 s -l),

a lower

the proportion

of bound

reaction

between

binding

of the

of both

low or high

monophasic

(at which

vesicles

kinetics

equation

strength.

equilibrium

only

provides

1, one can calculate

M ionic

f 0.14

equilibrium

strength

value

the

0.3 x lo3 M

that

rapid

M ionic

k

Using

is

to determine

% free

reflect

M, k = 1.54

fact

rates

At either

in DMPH4 up to a concentration

of the

possible

at any ionic

there

At 0.06

strength.

0.1 M, k = 2.8~

rates

each effect.

reduction

of reduction

the

reduction

to isolate

the actual

0.03

strength,

of cytochrome

makes it

rates

RESEARCH COMMUNICATIONS

and the

of ionic

(above

29 s -1 .

is

is

(below

means that

free

observed

difficult

observed

free

again

c, it

independent

M and 0.1 M ionic

observed

vesicles

the

completely

0.03

c to the

DMPH4, however,

c are

the completely

AND BIOPHYSICAL

this

can be estimated a surface

values

sites

is

1.6 x 10 -6 M.

case,

as follows:

area

258

is

the concentration

can presently

binding

[v],

c,

the concentration of bound [cl,

and

[C-VI

be obtained

for

[VI,.

the

of

same as the

total

cyto-

The maximum concentration since

a single

of 72 AL in a membrane

(15),

phosphoa phospho-

2:

Vol. 84, No. 1, 1978

lipid

concentration

total

external

the

of 2.02 surface

dimensions

minimum

BIOCHEMICAL

obtained

using

a value

of 20

uM is

Although

determining

the

accurate

c than

c are

the for

rate

free

the conformation

concept

that

interactions

would

a value

From

require

of 4.8

concentration

sites

vesicles.

x 10 18 binding

of 4.2

we are

of binding

interesting

for

sites

per

KD is

sites,

binding

while

site

constant

in the process

a

nM for

of binding

in asolectin

and unexpected

of reduction cytochrome

cytochrome as well

This

could

for

the

of experimentally

vesicles

c, making

it

to phospholipid

as electrostatic.

It

the heme crevice for

of cytochrome

the effect

of

for

mean that

up the heme crevice. c binding

the reason

findings

by DMPH4 was faster

c.

weakens

DMPH4 reduction

site

the dissociation

close,

4.4 x 10 21 ;2

for

a more

of ~~

by opening

phospholipid

to determine

for

of ferricytochrome

by DMPH4, e.g.

binding

the maximum estimate

limits

fairly

offer bilayer

strength,

of the

using

concentration

One of the most was that

estimate

would

unilamellar

a single

M ionic

these

determination

for

a maximum number

At 0.06

obtained

of cytochrome

liter

c (16),

the minimum

concentration. binding

per

giving

or 6.9 x 10 -6 M.

liter

with

area

thus

mg/ml)

x 10 -4 M (0.18

of cytochrome

of 1050 i2,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

these

vesicle

binding

more This

experiments bound

cytochrome

to vesicles

susceptible lends

to reduction

support

vesicles

involves

has been suggested

alters

that must

to the hydrophobic interaction

(17).

Further

work

be done

of vesicle

binding

on the kinetics

of

c.

ACKNOWLEDGMENT This investigation was supported GM 00249 to J.E.E. and NIH Research of General Medical Sciences.

by NIH Research Career Development Award Grant GM 18648 from the National Institute

REFERENCES 1. 2. 3. 4. 5.

Nicholls, P. (1974) Biochim. Biophys. Acta. 346, 261-310. Steinemann, A., and auger, P. (1971) J. Membrane Biol. 4, 74-86. Kimelberg, H.K., and Lee, C.P. (1969) Biochem. Biophys. Res. Comm. 2, 784-790. Papahadjopoulos, D., Moscarello, M., Eylar, E.H., and Isac, T. (1975) Biochim. Biophys. Acta 401, 317-335. Letellier, L., and Schechter, E. (1973) Eur. J. Biochem. 40, 507-512.

259

Vol. 84, No. 1, 1978

6. 7.

8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Quinn, P.J., and Dawson, R.M.C. (1969) Biochem. J. 115, 65-75. Bailey, D.N. (1974) J. Chem. Ed. 1, 488-489. Fiske, C.H., and Subbarow, Y. (1925) J. Biol. Chem. 66, 375-400. Greenwood, C., and Palmer, G. (1965) J. Biol. Chem. 240, 3660-3663. Goldkorn, T., and Schejter, A. (1977) FEBS Lett. 11, 44-46. Nicholls, P., and Malviya, A.N. (1973) Biochem. Sot. Trans. 1, 372-375. Letters, R. (1964) Biochem. J. E, 313-316. Whiteley, J-M., and Huennekens, F.M. (1967) Biochem. 2, 2620-2625. Barlow, G.H., and Margoliash, E. (1966) J. Biol. Chem. 241, 1473-1477. Johnson, S.M., and Bangham, A.D. (1969) Biochim. Biophys. Acta 193, 82-91. Dickerson, R.E., et al. (1971) J. Biol. Chem. 246, 1511-1535. Ivanetich, K.M., Henderson, J.J., and Kaminsky, L.S. (1974) Biochem. 2, 1469-1476.

260