Light-induced change in the buffer capacity of spinach chloroplast suspensions

Vol. 36, No. 4, 1969

LIGHT-INDUCED

BIOCHEMICAL

AND BKWHYSICAL RESEARCH COMMUNICATIONS

CHANGE IN THE BUFFER CAPACITY OF SPINACH CHLOROPLAST SUSPENSIONS G. M. Polya

Biology

ReceivedJuly

Division,

and A. T. Jagendorf

Cornell

University,

Ithaca,

N. Y.

14850

14, 1969

Summary: Illumination of spinach chloroplast suspensions in the presence of a redox dye causes large changes in the buffer capacity of the suspensions. The light-induced changes of buffer capacity above pH 6.4 can be completely reversed in the dark, while buffer capacity changes at pH values less than 6.4 While the light-induced buffer capacity are partly or completely irreversible. change at pH 7.2 and light-induced net proton uptake show similar responses to uncouplers and similar light requirements, the buffer capacity change at pH 6.2 has a much lower light intensity requirement than the other phenomena. The demonstration of light-induced buffer capacity changes in chloroplast suspensions calls for quantitative reappraisal of the literature dealing with light-induced net proton uptake by chloroplasts. Isolated

chloroplasts

take

of electron

transport,

Hind,

Neumann andJagendozf

1963;

sidered

as evidence

rylation

are

those

Jagendorf, dark these the

especially

1966a;

(Jagendorf

theory

have

one proton

available,

taken

of the up for

Accordingly,

attempts

to correlate

these

urements

in these

measured

by a glass

have with

electrode

evidence

photophospho-

see also

supporting

Jagendorf

this 1962;

gradient

alternative

and

concept Hind

in the

explanations testing

and

for

predictions

from

sought.

electron

model

is

passing

electron

have usually

rates

transport

of a stoichiometry a coupling

flux.

change

of

site.

of net proton

been of the rate

or by the absorbance

696

that

through

been made to measure

concomitant

experiments

1967;

for

(Shen and Shen,

However,

chemiosmotic

every

theory

1966,

conditions

has come to be con-

by an acid-base

and critical

been

phenomenon

phenomena

driven

under

pH 5 to 7 (Jagendorf

chemiosmotic

Other

1966b).

therefore

One prediction

This

ATP formation

and ATP synthesis

are

illuminated from

(Mitchell,

1967).

and Uribe,

phenomena

the

by Mitchell

of post-illuminatton 1963)

, 1964). with

Jagendorf,

when

in the region

consistent

as elaborated

and Uribe,

up protons

uptake

The primary of change of a dye.

and meas-

of pH as These

pH

Vol. 36, No. 4, 1969

changes

are

titration

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

translated

curve

below

dark

is

appreciable

proton

quantitative

in

significance

changes

in a water-cooled

using

electrode

a Moseley

Autograf

by a Sylvania Light

meter.

Titrations

the suspension capacity ition

determined Fig.

which

it

is

apparent buffer

vicinity ive

the buf:Eer

Curves

are

of pH 7; a large

number

of these

was provided

and a Corning

2418 red

Model

65 Radio-

commenced when the

turning

on the

from

the

Light-induced

buffer

pH of

light.

Buffer

pH shift

on add-

proton

pH shift

capacity

shown for

uptake

was

by means of titration

as a function

the buffer

after

the

large

capacities

light

changes

of the pH at in the dark

has been

turned

in the buffer

of experiments It

phenomena.

have is

as readily

in the vicinity

697

clear

confirmed that

of pH 7.

Fig.

in the

the qualitat-

the buffer

reversible

capacity

in the dark lb

off.

capacity.

has a maximum at pH 6 and a shoulder

of pH 6 is not change

Light

of illumination.

causes

curve

capacity

after estimated

and in the dark

capacity

:in the vicinity

value

were

using

and recorded

a YSl-Kettering

light-induced

illumination

reproducibility

change

a steady

suspension

light,

that

using

chloroplasts

same conditions

la shows the

in the

The light

traps

steady-state

is measured.

initially,

water

measured

(prepared monitored

10 pH meter,

through

on the

Chloroplast

were

recorder.

and

from market

NaOH solutions

chart

of NaOH solution.

in the

1963).

pH changes Model

was routinely

of a known aliquot

curves

prepared

and Jagendorf,

in the

a pH change

shed uncertainty

7100B strip

of illuminated

determined

in 10 mM NaCl were

prepared

presented

of chloroplast

ratios.

vessel.

were

suspension

results

to the

results

of tile'

freshly

Model

intensities

from the

between

and a Corning

had reached

of the

quantitated

It

with

Sun Gun and filtered

filter.

to the relation

(Hind

H20) glass

curve

suspended

with

a Corning

a titration

determinations

titrated

our

capacity

of past

previously

by reference

the buffer

These

were

boiled

in

so that

suspensions

uptake However

the light.

chloroplasts

as described

proton

suspension.

applicable

uptake

Swollen spinach

of net

due to illumination,

not neccessarily

the net

units

of the chloroplast

demonstrate

spspensions

into

as is

shows the result

Vol. 36, No. 4, 1969

BIOCHEMICAL AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

0.6 0.5

z 3 I,

0.3

L0’ 0.2 .g : 3 F:

0.1 0.4 I

(a)Buffer capacity of a suspension of chloroplasts (125 Pg chl/ml) Figure 1. in 10 mM NaCl-10 uM pyocyanine (total vol. 4 ml) at 5.5', determined by titration in the dark before illumination (DARK 1, w), when illuminated with red light (21.5 x lo5 ergs cme2 set-l) (LIGHT, o), and from 7 mins after the cessation of illumination (DARK 2, A). The chloroplast suspension used to determine all 3 curves was illuminated for a total of 31 mins. @)Buffer capacity of chloroplast suspensions (125pg chl/ml) in 10 + NaCl-25 PM pyocyanine at 5.5O, titrated in the dark (DARK 1, e), in the dark from 5 mins after a prior 5 min illumination with red light (10.3 x lo5 ergs cmm2 set-l) (DARK 2, A), and while illuminated with red light (10.3 x lo5 ergs cm-2 set-1) (LIGHT, 0).

of a similar

experiment

of exposure

of the chldroplast

reversibility 8.2 while

in which

the

of the

buffer

buffer

capacity

a lower

light

suspension capacity

to light

change

change

intensity

wer

and a shorter

resulted the range

in the vicinity

time

in a complete of pH 6.4

to

of pH 6 was again

pH

largely

irreversible. Other linear that

experiments

function the

chloroplasts.

lines

have

of chloroplast extrapolate This

rules

shown that

the buffer

concentration to the

same buffer

out a contribution

capacity

in the capacity of added

light

at pH 7.2 is and in the dark

in the absence redox

a and

of

dye to the extent

Vol. 36, No. 4, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

0 LIGHT

INTENSITY

2 cm-*

(ergs

3 see-’

7

7.4

x 10-5)

Figure 2. Light intensity dependence of the light-induced buffer capacity changes at pH 6.2 (0) and pH 7.2 (e), and the light-induced net proton uptake (initial pH 6.20 f 0.04) (A), determined for chloroplast suspensions (125 pg chl/ml) in 10 mM NaCl-25 ~11 phenazine methosulfate (total vol. 4 ml) at Curves describing buffer capacity in the light and in the dark as functions 5.s". of pH were determined for all experimental situations and the buffer capacity changes at pH 6.2 and pH 7.2 determined by interpolation.

of the buffer Fig. 6.2,

were

intensitfes ured

with

change

2 shows the

of the change

capacity data

capacity

at pH 6.2 obtained

light.

intensity

dependence

in buffer

capacity

at pH 7.2,

in the using for

2 different buffer

in the

light

required

half-maximal

at pH 7.2

presence

capacity

are

approximately

the

for

half-maximal

change

as cofactor.

half-maximal

change

same, but in buffer

is clear

capacity

699

I lists

of these that

the

at pH 7.2 and net

considerably

uptake

greater at pH 6.2.

pH

Similar the light

3 phenomena,

meas-

intensities

for

light proton

than

at

in buffer

methosulfate.

Table

saturation It

proton

and of the change

of 25 uM phenazine

pyocyanine

cofactors.

of net

that

uptake

at pH 6.2

required

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 36, No. 4, 1969

TABLE LIGHT

INTENSITY

I

DEPENDENCE OF NET PROTON UPTAKE AND BUFFER CAPACITY CHANGE

The data 2 separate

from

were obtained experiments.

as described

in the

Phenomenon

legend

Light

Cofactor:

25 nM phenazine

(2)

methosulfate

Buffer

capacity

change

(pH 7.2)

0.29

Buffer

capacity

change

(pH 6.2)

0.04

Net proton

uptake

Cofactor:

(initial

pH 6.20

k 0.04)

0.33

25 uM pyocyanine

Buffer

capacity

change

(pH 7.2)

0.69

Buffer

capacity

change

(pH 6.2)

0.10

Net proton

uptake

Effects

(at

shown

II.

pH of 6.25)

capacity

change

pH 6.16

on net

in Table

an initial

of the buffer capacity

(initial

of uncouplers

at pH 7.2 are uptake

2 and derive

intensity for half-maximal effect cm-2 set-1 x 10-5)

(ergs (1)

to Fig.

change

* 0.04)

proton

uptake

and on buffer

capacity

change

The inhibition

of light-induced

net

is qualitatively

parallelled

by inhibition

at pH 7.2.

in the vicinity

0.63

Effects

of pH 6 are

of uncouplers

quite

complex

proton

on the buffer

and will

be describ-

predictable

as an

ed elsewhere. These inevitable with

pH-dependent consequence

pH; the

thing.

This

chloroplasts

its

is best

(pHD),

termination

in buffer

of a light-induced

two types

in the

pH in the dark (A),

changes

of pkeaomena illustrated

light the being

and in

proton

are

simply

by reference the dark

light-induced a point

capacity

(pHL) 700

on the

uptake

whose

different to the

shown pH rise

were

aspects titration

in Fig.

3.

is given light

extent

of the same curves

Starting

by the vertical

titration

varies

curve.

for with

any line Altern-

Vol. 36, No. 4, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE II EFFECTS ON UNCOUPLERS ON LIGHT-INDUCED

NET PROTON UPTAKE AND BUFFER

CAPACITY CHANGE IN SPINACH CHLOROPLAST SUSPENSIONS

Buffer capacity changes as functions of pH were determined for suspensions of chloroplasts (125 pg chlorophyll/ml) in 10 mM NaCl-25 PM pyocyanine at 5.5" in each of the experimental situations and the buffer capacity change at pH 7.2 determined by interpolation, Net proton uptake (initial pH 6.25 * 0.03) was quantitated from the extent of the steady-state pH rise and the titration curves of the suspensions in each experimental situation, determined in the same conditions of illumination (10.3 x 105 ergs cm-2 set-1 in all cases). Treatment

1.

2.

Net Proton (pequiv/mg

Buffer Capacity Change (pH 7.2) @equiv/pH/mg chl)

Control 0.25 mM chlorpromazine 1 mM NH4Cl

0.718 0.136 0.552

0.116

1% 1% 1% 1% 1%

0.644 0.472 0,348

0.448 0.232 0.164 0.062 0.072

EtOH EtOH-5CIM CCCP EtOH-1OCIM CCCP EtOH-25VM CCCP EtOH-50CIM CCCP

atively,

the

curves

indicates

constant light

intercept

at that

PH.

buffer

derivatives

has been

models

from

and does not

(B) between to keep

on,

i.e.

of the

the two titration

the pH of the

the net

proton

light-induced

defines

the light

in turn

simply

uptake

in the

pH rise,

super-

titration obtain

from

suspension

curve. the

The

first

curves.

change

or a combination

neccessarily

turned

curves

titration

light-induced

required

curve

capacity

of these

line

pa-dependence

titration

The light-induced 2 extreme

of acid

Thus the

on the dark

pH-dependent

0.134

on the horizontal

light

0,412 -0.058

0.036

the amount

once the

imposed

arises

Uptake chl)

in buffer

capacity

can be explained

One major

of both.

conformational

changes

involve

translocation

proton

701

possibility

of membrane across

in is

terms that

polyelectrolytes the

thylakoid

of it

Vol. 36, No. 4, 1969

BiOCHEMlCAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

6.5

6.0 0

0.2

0.3

p equiv

0.4

0.5

0.6

ADDED

OH-

of titration curves of chloroplast suspensions (125 Figure 3. Portions chl/ml) in 30 mM NaCl-10 PM pyocyanine (total vol. 4 ml), determined at in the dark (A), or 2 when the suspension was illuminated with red light (21.5 x lo5 ergs cm' se4) (0). Also shown is the superimposition the dark titration curve of the light-induced pH rise (a), determined the same experimental conditions. The quantitation of the light-induced equivalents clearly derives from line ) in terms of proton ine (C).

membrane.

The alternative

across

thylakoid

the

equivalence model

The fact consequences If

the net

and Jagendorf, state

extent

ever,

if

the

that with

is

membrane

of an active

can produce

take.

view

in the

proton results

proton

1964;

the steady-state

light

uptake

is

extent

titrated

of protons

is

through

in the

attained

efflux.

uptake

light

Either

uptake

proton

pH in the

and Fiat,

can be measured

proton

has important

light-induced

at constant Tyszkiewicz

702

translocation

proton

to quantitate

of the

on in pH (B)

here. increases

Girault,

of proton

a net

and a passive

capacity

to attempts

is

and equilibrium

demonstrated

Galmiche,

and kinetics

there

influx

the buffer respect

that

yg 5.5"

is

1967)

with calculated

light the

validity. from

up(Neumann steadyHow-

Vol. 36, No. 4, 1969

primary

recordings

pH change

in

perimental

of pH change,

terms

of proton

conditions

quisite

calculations

kinetics how rapidly

the

based

have

of proton

flux

the buffer

actual

on dark

to estimate

clearly

net

capacity

not

to quantitate

case of attempts

is changing

of such re-

that if

such results

in fact

they

are

derive

The extent

of the error

to measure

the

recordings, after

ex-

in any of the pub-

indicate

curves.

time

The details

movement

the

in identical

been specified

proton

titration

pH vs.

titration

etc.).

Our data

in the from

the

must be performed

of illumination

titrations

by minimizing

more difficult

then

of such experiments.

reports

in error

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

equivalents

(i.e.

quantitating

lished

from

BIOCHEMICAL

because the

light

it is

is

initial

is not turned

clear on or

off.

ACKNOWLEDGMENTS Supported in part by Grant GM-14479 from the National Institutes of Health, by a Kettering Award, and by a C.S.I.R.O. Postgraduate Studentship to G. M. Polya. Technical assistance was provided by Nancy Killilea and Mrs. I. 0~01s.

REFERENCES Galmiche, J. M., Girault, G., Tyszkiewicz, E., and Fiat, R. (1967), C. R. Acad. Sc,i., Paris 265, 374. Hind, G., and Jagendorf, A. T. (1963), Proc. Nat. Acad. Sci. 49, 715. Jagendor.E, A. T. (1967), Fed. Proc. 26, 1361. Nat. Acad. Sci., Nat. Res. Council, Jagendorf , A. T., and Hind, G. (1963), Publ. No. 1145, 599. JagendorE, A. T., and Uribe, E. (1966a), Brookhaven Symp. in Biol. l9, 215. JagendorE, A. T., and Uribe, E. (1966b), Proc. Nat. Acad. of Sci. 55, 170. Mitchell, P. (1966), Biol. Rev. 4l, 445. Mitchell, P. (1967), Fed. Proc. 26, 1370. Neumann, J., and Jagendorf, A. T. (1964), Arch. Biochem. Biophys. 107, 109. Shen, Y. K., and Shen, G. M. (1962), Scientia Sinica 11, 1097.

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