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THE EFFECTS OF UNCOUPLERS ON THE INTENSITY AND DURATION
OF DELAYED FLUORESCENCE AND ON THE RATE OF ELECTRON
TRANSPORT IN ISOLATED CHLOROPLASTS

BY

Duncan H. Bell

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1979

ABSTRACT
THE EFFECTS OF UNCOUPLERS ON THE INTENSITY AND DURATION

OF DELAYED FLUORESCENCE AND ON THE RATE OF ELECTRON
TRANSPORT IN ISOLATED CHLOROPLASTS

BY

Duncan H. Bell

Information is presented in this thesis about the
effects of electron transport uncouplers, electron transport
inhibitors, and rates of electron transport on the delayed
fluorescence from spinach chloroplast lamellae. A laser
phosphorOSCOpe was combined with a spectrophotometric device
to permit simultaneous determinations of microsecond delayed
fluorescence and noncyclic electron transport under nearly
steady-state conditions of most of the electron transport-
generated transmembrane electrochemical gradients.

Uncouplers of photOphosphorylation and the presence
of phosphorylation conditions increased the intensity of
that part of the delayed fluorescence observed less than
100 us after light extinction but inhibited that part of the
delayed fluorescence observed after longer delays. This
initial increase in delayed fluorescence implies that

electrochemical gradients (which are decreased or abolished

Duncan Hadley Bell
under such conditions) are not important factors contrib-
uting to the back—reactions responsible for the delayed
fluorescence.

The initial intensities of the delayed fluorescence
at pH's above neutrality were well correlated with the
rates of electron transport when the rate-limiting steps
in transport between the two photosystems were increased
by uncouplers or decreased by the inhibitors diuron or
cyanide. On the other hand, inhibitors of electron trans-
port on the oxidizing side of photosystem II did not in-
hibit delayed fluorescence nearly as severely as they
inhibited prompt fluorescence and, therefore, it seems
probable that the chemical back-reactions responsible for
delayed fluorescence actually increased. Presumably, dif-
ferent rates of electron transport alter the amount of
oxidized reaction center chlorOphyll P680 and the amount of
reduced electron acceptor (Q) formed in the light and, in
so doing, alter their initial rate of back-reaction and
enhance the intensity of the delayed fluorescence.

The decay of delayed fluorescence was not a simple
exponential function but consisted of two or more phases.
The rate of decay of the delayed fluorescence was unaltered
by the presence of uncouplers during the first 100 us after
light extinction. Thereafter the decay was much faster in
the presence of uncouplers than in their absence. Thus the
residual delayed fluorescence after 100 us had a half-life of

about 800 us in the absence of uncouplers but a half-life of

Duncan Hadley Bell

300—500 us in the presence of uncouplers. These observa-
tions probably can be explained in terms of the known
increase in the rate of oxidation of plastoquinone in the
presence of uncouplers, since an increase in the rate of
this oxidation might begin to affect the level of Q- within
a few hundred microseconds.

In this thesis are also described the effects of a wide
range of uncouplers on phosphorylation and on electron trans-
port at pH values ranging from 6.5 to 9.0. Large increases
in the rates of electron transport with little if any in-
hibition of phosphorylation were found near neutrality when

the pK's of the uncoupling amines were also near 7.0.

To my wife, Sally

ACKNOWLEDGMENTS

I wish to thank the many people who have assisted in
the preparation of this thesis by their constant support,
timely encouragment and wise advice.

Some of these friends deserve special recognition:

My father — for the many years of support for my
scientific endeavors and for allowing
me to develop skills with tools and
machines

Jim Perley - for starting me down the paths of plant
physiology

Norman Good- for five years of friendliness, challenge,
advice and fruit

Alfred Haug- for cheerful encouragment and for the use
of the laser phosphoroscope

Ken Nadler, Ken Poff, and Gerry Babcock -
for serving on my guidance committee

Sarah Hitchcock Bell - for constant love, companionship,
pleasant distractions, and for drawing

most of the figures

iii

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . . . . . . . .

LIST OF FIGURES . . . . . . . . . . . . . . . . . .

LIST OF ABBREVIATIONS . . . . . . . . . . . . . . .
SECTION I

THE EFFECTS OF UNCOUPLERS AND ELECTRON TRANSPORT
INHIBITORS ON MICROSECOND-DELAYED FLUORESCENCE

INTRODUCTION 0 O O O O O O O O O O O C O O O O O O

Recombination Hypothesis . . . . . . . . . . . .
Triplet State Hypothesis . . . . . . . . . . . .
Semiconductor Hypothesis . . . . . . . . . . . .
Relation to Photosystem II . . . . . . . . . . .
Lavorel Model . . . . . . . . . . . . . . . . . .
The Importance of Delayed Fluorescence . . . . .
Techniques for Measuring Delayed

Fluorescence . . . . . . . . . . . . . . . . .
Effects of Uncouplers and the Importance of

the High Energy State . . . . . . . . . . . . .
Crofts-Fleischman Hypothesis . . . . . . . . . .

METHODS O O O O O O C O I O O O O O O O O O O O O 0

Isolation of Chloroplasts . .
Hydroxylamine Treatment . .
Cyanide Treatment . . . . . .
Laser Phosphoroscope . . . .

RESULTS I O O O O O O O O O O O O O I C O O O O O 0
DISCUSSION 0 O C O O O O O O O O O O O O O O O O 0

Initial Intensity of Delayed Fluorescence . . . .
Decay of Delayed Fluorescence . . . . . . . . . .

iv

Page
vi
vii

ix

\lChUI-L‘UDN H

13
14
20
20
22
23
24
28
40

40
46

SECTION II

THE EFFECTS OF AMINES AND OTHER UNCOUPLERS ON
ELECTRON TRANSPORT, ATP SYNTHESIS, AND P/E2
Page

INTRODUCTIOIJ O O O O O O O O O O O O O O O O O O O O 52

Electron Transport Generates a

pH Gradient . . . . . . . . . . . . . . . . . . . 53
A pH Gradient Generates AT . . . . . . . . . . . 54
Uncoupling and the Control of the Rate of

Electron Transport . . . . . . . . . . . . . . . 55
Uncoupling by Amines . . . . . . . . . . . . . 57
Uncoupling by Weak Acids . . . . . . . . . . . . . 58
Uncoupling by Ionophores . . . . . . . . . . . 59
Uncoupling by Quasiionophores . . . . . . . . 59
Unconventional Uncoupling. . . . . . . . . . . . . 60

METHODS . . . . . . . . . . . . . . . . . . . . . . 62
RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 64
LITERATURE CITED . . . . . . . . . . . . . . . . . . 84
APPENDICES . . . . . . . . . . . . . . . . . . . . . 94
Appendix I: Stimulation of Microsecond-delayed
fluorescence from Spinach Chloroplasts by

Uncouplers and by Phosphorylation . . . . . 94

Appendix II: Energy Transfer Inhibition by
Pyrophosphate . . . . . . . . . . . . . . . 104

Appendix III: The Suitability of Several New
Aminosulfonic Acid Buffers in Studies of
Photosynthesis . . . . . . . . . . . . . . 108

Appendix IV: The Design of an Inexpensive,
Reliable, and Rapid-acting Fraction
COlleCtor. o o o o o o o o o o o o o o o o 111

Table

II.

III.

LIST OF TABLES

Page

Effects of an Inhibition of Electron
Transport on Delayed Fluorescence when

the Inhibition is on the Oxidizing Side

of Photosystem II . . . . . . . . . . . . . .

Correlations Between the Stimulation of
Electron Transport and the Inhibition

of Phosphorylation by Uncouplers at

pH 6.5. O O O O O O O O O O O I O O O O O O O

A Survey of the Effectiveness of Amines
with pK's near Neutrality in Stimulating
Electron Transport at pH 6.5 . . . . . . . .

APPENDIX I

The Effects of Ferricyanide, Uncouplers,

and DCMU on the Relative Intensities of

5-10 microsecond-Delayed Fluorescence

and Electron Transport . . . . . . . . . . .

APPENDIX III
Rates of Electron Transport and ATP Synthesis

in Illuminated Spinach Chloroplast
Lamellae with Various Buffers . . . . . . . .

vi

37

78

82

100

110

LIST OF FIGURES

Figure

l.

Delayed Fluorescence in Spinach
Chloroplasts in the Presence and
Absence of the Uncouplers NH Cl,
Nigericin, Methylamine-HCl, 3nd
Gramicidin . . . . . . . . . . . . . . .
Delayed Fluorescence in Spinach Chloro-
plasts in the Presence and Absence

Of ADP and Orthophosphate . . . . . . .

The Effect of the Electron Trans-

port Inhibitor DCMU on the 5-10 us
Intensity of Delayed Fluorescence

in the Presence and Absence of
Gramicidin . . . . . . . . . . . . . . .

The Rates of Electron Transport and
the 5-10 us Delayed Fluorescence In-
tensity at pH's from 6.5 to 9.0 in

the presence and Absence of Uncouplers

Effects of Uncouplers on Electron
Transport and Photophosphorylation
at pH's from 6.5 to 9.0 . . . . . . . .

The Effects of Light Intensity on
the Uncoupling by Amines . . . . . . . .

APPENDIX I

Delayed Fluorescence in Spinach Chloro-
plasts and Its Rate of Decay in the
Presence and Absence of Uncouplers . . .

Delayed Fluorescence in Spinach Clhoro-
plasts and Its Rate of Decay in the
Presence and Absence of ADP and
Orthophosphate . . . . . . . . . . . . .

The Relationship of Electron Transport
to the Intensity of Delayed Fluorescence

vii

Page

29

31

34

.65

-80

.99

.99

. 100

Figure Page

4. Delayed Fluorescence and Electron
Transport as Functions of the Ex-
citing Light Intensity in the
Presence and Absence of the Uncoup-
ler Gramicidin . . . . . . . . . . . . . . . 101

APPENDIX II

1. Effects of Pyrophosphate on the Rates of
Phosphorylation and Electron Trans-
port in Spinach Lamellae . . . . . . . . . 105

2. Double Reciprocal Plot of the Inhi-
bition of Phosphorylation by Different
Levels of Pyrophosphate at Various
Concentration of ADP . . . . . . . . . . . . 107

APPENDIX IV
1. Diagram of the Escapement Mechanism
Which Regulates the Advancement of
the Fraction Collector . . . . . . . . . . . 112

2. Schematic Diagram of the Fraction
Collector Circuit . . . . . . . . . . . . . . 115

viii

BCD
CCCP
Chl
DCMU
DF
DF*
DMAP
GD
HEM

HEPES

LED

MOPS
P/e2

P680

PF
PS I

PS II

TAPS

LIST OF ABBREVIATIONS
Binary-coded decimal
carbonylcyanide 3-chlorophenyl hydrazone
chlorophyll
3-(3,4-dichlorophenyl)-l,l-dimethylurea
uncorrected counts of delayed fluorescence
corrected counts of delayed fluorescence
3-dimethylaminopropionitrile
gramicidin
N-B-hydroxyethylmorpholine

N-hydroxyethylpiperazine-N'-ethanesulfonic
acid

intensity of the light absorbed

flux of excitons injected into the antenna
chlorOphyll as a result of the back-reactions
that give rise to delayed fluorescence
light-emitting diode
3-(N-morpholino)propanesulfonic acid
orthophosphate (PO4-3)

phosphorylation efficiency as moles of ATP
formed per pair of electrons

reaction center chlorophyll associated with
photosystem II

counts of prompt fluorescence
photosystem I

photosystem II

primary acceptor of photosystem II

3-tris(hydroxymethyl)-methyl-3-aminopr0pane
sulfonic acid

first secondary electron donor of photosystem II
ix

SECTION I

THE EFFECTS OF UNCOUPLERS AND ELECTRON TRANSPORT

INHIBITORS ON MICROSECOND-DELAYED FLUORESCENCE

INTRODUCTION

Delayed fluorescence was discovered by Strehler and
Arnold (1951) in chloroplasts. They were performing a fire-
fly extract assay for the determination of ATP in these
chloroplasts when they noticed that even the controls which
omitted the firefly extract exhibited a delayed fluorescence
that persisted for several minutes. Early experiments were
quick to establish that the phenomenon is related to the
process of photosynthesis. It was found that the delayed
fluorescence (also called luminescence or delayed light
emission) occurs in photosynthetic systems - be they leaves,
algae, chlorOplasts, or photosynthetic bacteria - and is
lacking in non-photosynthetic systems. The action spectrum
for delayed fluorescence was found to parallel closely that
for photosynthesis in green (Strehler and Arnold, 1951),
bluegreen, and red algae as well as photosynthetic bacteria
(Arnold and Thompson, 1956). Heat treatment of chloroplasts
(Strehler and Arnold, 1951) and experiments with Mn-deficient
mutants of algae (Kessler et al., 1957) also suggested that
at least part of the photosynthetic apparatus must be
functionally intact to permit delayed fluorescence.

Emission spectra for both delayed fluorescence and
prompt fluorescence are very similar in many organisms
(Strehler and Arnold, 1951; Arnold and Thompson, 1956;

Clayton, 1965), suggesting that the light emitted as delayed
1

fluorescence is due to the return to the ground state of
chlorophyll molecules in the lowest excited singlet state
and is not the result of the phosphorescence of some long-
lived triplet state which would emit quanta at longer wave-
lengths than those observed for delayed fluorescence. (But

see below.)

Recombination Hypothesis

 

To explain this phenomenon, Arthur and Strehler (1957)
proposed what has come to be called the recombination hypothe-
sis (see Lavorel, 1975a). Its major tenets are these: a)
Delayed fluorescence results from a back-reaction (recombina-
tion) of the primary photOproducts of photosynthesis. In
fact, such an occurrence can be induced chemically in the
dark by first oxidizing and then reducing chlorophyll a
dissolved in organic solvents (Goedheer and Vegt, 1962).

b) The emissions at times longer than the times required
for immediate back-reactions are made possible by the
reversal of the slower electron transfer steps associated
with some dark reduction of Q by the electron carriers on
the reducing side of photosystem II and some dark oxidation
of the reaction center chloroPhyll P680 by some electron
carriers on the oxidizing side of photosystem II. c) The
electron from the reduced acceptor is injected into the
oxidized chlorophyll molecule forming an excited singlet

state exciton. This exciton migrates into the antenna

chlorophylls where it may decay with the production of a
quantum of light. d) Fluorescence and delayed fluorescent-
excitons are not distinguishable. e) The excitons are
subject to radiative or non-radiative decays, migration,
and trapping by "Open" centers (That is, by doing the photo-

chemical act of charge separation to give 0- and P680+).

Triplet State Hypothesis

 

Investigations with organic molecules in fluids and
crystals (Priestley and Haug, 1968), and with chlorophyll
in_zi£rg (Parker and Joyce, 1966) have suggested that
delayed fluorescence might involve the triplet state in-
directly through intersystem crossing back to the singlet
state (see Malkin, 1977). The likeliest mechanisms are
triplet—triplet annihilation (where two triplet excitons
combine to form one singlet exciton), absorption of a
quantum of light by a chlorOphyll molecule in the triplet
state, or thermochemical excitation of the triplet chloro-
phyll to the singlet (E-type emission as demonstrated with
eosin. Parker, 1968). The second and third approaches
raise the triplet chlorophyll to a higher triplet state
which may then be converted to a singlet state and then
fluoresce. It should be pointed out, however, that detection
of the triplet state in chloroplasts or whole organisms is
by no means a simple task (especially at room temperatures)

and although there are a few reports of its existence

(Uphaus et al., 1974; Leigh and Dutton, 1974; Haberkorn

and Michel-Beyerle, 1977), theories invoking its involve—
ment in the delayed fluorescence of photosynthetic organisms
(Stacy et al., 1971) seem less tenable than the recombination

theory.

Semiconductor Hypothesis

 

Several lines of evidence have led to a semiconductor
explanation for the phenomena of delayed fluorescence.
Tollin et a1. (1975) observed delayed fluorescence at an
extremely low temperature (77°K) which forced them to rule
out the participation of any enzymatic process. Also,
experiments by several laboratories (Arnold and Sherwood,
1975; Arnold and Azzi, 1968; Ichikawa et al., 1975 Lurie
and Bertsch, 1974) determined that algae, leaves, and chloro—
plasts could give rise to‘thermoluminescence. This is
light (fluorescence) emitted in the dark at specific tempera—
tures by material that is slowly warmed after having been
frozen to 77°K during illumination. Bertsch and his co-
workers (Bertsch and Lurie, 1971; Bertsch et al., 1971)
proposed a two-quantum electron-hole model to account for
thermoluminescence and for the stimulation by artificial
electron donors of the delayed fluorescence associated with
Tris-treated chloroplasts. In his model, delayed fluore-
scence arises within an aggregate of chlorOphyll molecules.

The absorption of two quanta create two electron-hole pairs.

In one of the pairs, the hole oxidizes a primary donor and
in the other pair, the electron reduces a primary acceptor.
This leaves an electron and a hole that are separated and
are free to migrate. There is a significant probability that
these mobile species will recombine before being trapped
again, and such an event could give a quantum of delayed
fluorescence.

Such a model should operate even at 77°K (Type 1). To
explain the two other phenomena (thermoluminescence and
tris-donor effects) two variations were proposed. One (Type
2) requires thermal activation of the resulting hole and
electron before they can migrate and the other (Type 3) re-

quires reverse electron flow to create the electron and hole.

Relation to Photosystem II

 

In higher plants, the majority of the delayed fluores-
cence detected seems to be emitted by the chlorophylls assoc-
iated with photosystem II. The action spectrum for delayed
fluorescence corresponds to the action spectrum for photo-
system II whereas light that stimulates PS I exclusively
(> 700nm) depresses the intensity of delayed fluorescence
(Goedheer, 1962). Mutants lacking photosystem II show
very little delayed fluorescence while mutants lacking only
photosystem I have normal delayed fluorescence (Bertsch and
Azzi, 1965; Haug et al., 1972). Subchloroplast particles

enriched in photosystem II produce more delayed fluorescence

than those enriched in photosystem I (Lurie et al., 1972).
It should be mentioned that there have been some re-

ports of delayed fluorescence arising from photosystem I

(Shuvalov, 1976) but its intensity is much less than that

associated with photosystem II.

Lavorel Model

 

Lavorel proposed in 1968 that the delayed fluorescence
intensity could be expressed in a manner analogous to the
fluorescence intensity. In the latter case, the relationship

is:

Where F is the fluorescence intensity, Q is the quantum yield
for fluorescence, and I is the intensity of the light absorbed.
In the case of delayed fluorescence, the relationship is:

L=<I>J.

Where L is the delayed fluorescence intensity and J is the
flux of excitons injected into the antenna chlorophyll com-
plex as a result of back-reactions in the photosystem II
reaction center. These two formulae have the quantum yield
term in common, reflecting the supposition that once an ex-
citon is formed - either by the light excitation of the re-
action center pigment via the antenna chlorOphyll or by the
reduction of the oxidized reaction center pigment by Q- - it

will disappear in a manner that is independent of its method

of creation. In other words, conditions which affect the
yield of prompt fluorescence (such as cations, pH gradients,
etc.) should affect the delayed fluorescence yield in a sim—
ilar manner.

As Clayton (1969) has pointed out, it is important to
consider which component of the fluorescence yield to use.
The yield of fluorescence is normally considered to have two
components. The so call "dead" fluorescence yield is an in-
variable portion of the fluorescence and is thought to be due
to those chlorOphyll molecules which are not in contact with
reaction centers. There is also a variable component of the
fluorescence yield, however, which responds to the condition
of the reaction center. It is this potentially variable
portion of the fluorescence which reflects the fate of the
singlet excitons responsible for both delayed fluorescence
and prompt fluorescence.

Certain difficulties with the Lavorel model arise how-
ever if the excitation energy can migrate freely between the
reaction centers. If this is the case, the yield per unit
is not only dependent upon the reaction center being Open or
closed, but also on the state of neighboring reaction centers
(the so called flat or "cluster" effect of Lavorel and Joliot,
1972). Under these circumstances the value of G for delayed
fluorescence and for prompt fluorescence may not be the same.

It should be remembered that J will depend upon the
competing reactions that lower the amounts of the reactant

species, Q‘ and P680+ available to back-react. Such reactions

8

are the oxidation of Q- by a secondary acceptor and the re-
duction of the oxidized chlorophyll by a secondary donor, Z.

In some models (Lavorel) the ZCth reaction center
complex is considered to be one unit. In this case, the
active species is Z+Ch10-, a photoelectric dipole. Lavorel's
notation however seems to minimize the importance of the
state of the reaction center chlorophyll. True, the
reduction of the pigment by Z (forming Z+) is one of the
fastest electron transfers under consideration (see discussion)
but it may become significant when one considers the delayed

fluorescence arising very soon after the light extinction.
The Importance of Delayed Fluorescence

It is not clear what function if any delayed fluores-
cence plays in the plant or in the chloroplast. The quantum
yield for delayed fluorescence is only about 10—4 (Zankel,
1971) and its role in photosynthesis must necessarily be
minimal. However, its role in photosynthesis research may
be considerable since it is a useful tool with which to
investigate the properties of photosystem II. It is im-
portant as a non-intrusive indicator of a variety of con-
ditions related to electron transport, ion fluxes, and ATP
synthesis.

The intensity of microsecond delayed fluorescence
varies with the same periodicity (with saturating flashes)

as the oxygen yield in chloroplasts and algae. This suggests

use of delayed fluorescence as another experimental tool to
probe the mechanism of oxygen liberation and the condition
of the so called "S states" (Kok et al., 1970). Also, as
will be shown, the millisecond delayed fluorescence seems to
reflect the energized state of thylakoid membranes and as
such may be employed to corroborate the values of membrane
potentials obtained by electrochromic shift determinations
(Barber, 1972).

As shown in this thesis, the microsecond delayed
fluorescence reflects the steady-state rates of electron
transport through photosystem II.

Finally, as considered in the discussion, the kinetics
of the decay of delayed fluorescence may provide information
about the rate constants of the separate electron transfer

reactions associated with photosystem II (Vierke, 1979).

Techniques for Measuring Delayed Fluorescence

 

Since much of the information about delayed fluores-
cence can only be interpreted when it is clear how the in-
formation was obtained, it is important to review briefly the
experimental techniques at an early point in this thesis.

There are only two components common to all delayed
fluorescence experiments - a light source and a light de-
tector. When one starts to add additional components (oscil-
loscopes, rotating and triggered shutters, stOpped-flow de-

vices, signal averagers) in addition to alterations in the

10

light sources (xenon lamps, xenon lasers, pulsed lasers) and
light detectors (photocells, photomultipliers, photon
counting) the explosion in different techniques (and hard-
ware) is astonishing. Moreover, all this is compounded by a
wide range of photosynthetic organisms studied (red, blue-
green, and green algae, bacterial chromatophores, frozen and
non—frozen chloroplasts, intact plants). Unfortunately, the
profusion of techniques has created a very diverse body of
phenomena - all of which are in some way related to delayed
fluorescence - but conclusions reached with one approach
often are not applicable to a different approach.

Since the discovery of delayed fluorescence, there
has been one dominant theme in the development of new
approaches. This theme can be identified by a single word,
"faster". In the twenty—eight years since delayed fluores-
cence was discovered, the minimum time for light measurement
has decreased from hundreds of milliseconds to a few micro-
seconds. This has been possible in large part because of
equally impressive reductions in the cut-off times of the
actinic light. Since the prompt fluorescence intensity is
three orders of magnitude greater than the delayed fluores-
cence intensity, any "tail" of the actinic light swamps the
delayed light with prompt fluorescent light and makes re-
laible determinations of delayed light impossible.

Nearly all delayed fluorescence experiments can be
divided into three groups; phosphoroscope, single flashes,

environment-triggered. These divisions are by no means

ll

clear-cut and combinations of all of the methods have been
reported.

The phosphoroscope method was first employed in the
study of the phosphorescence of organic molecules. With the
"Becquerel" type of phosphoroscope, the sample is placed
between two rotating sectors with Openings at a fixed angular
distance. The decay of delayed fluorescence after the ac-
tinic light pulse is determined by altering this angular
distance or by varying the speed of rotation of the sectors.
The minimum time after light extinction is about 0.1 ms with
this technique, the limitations being the width of the beams
being chOpped, the width of the chOpping windows, and the
speed of the rotating sector.

Lavorel (1971) and Haug et a1. (1972) have modified
the phosphoroscope approach by using a laser as the light
source. Due to the parallel nature of the light beam
produced, a converging lense can focus the beam almost to a
point, the chopping of which by a high-speed rotating sector
gives extinction times of less than 250 ns (Haug et al., 1972).

The phosphoroscope technique however has the possible
disadvantage that, since the delay of delayed fluorescence
lasts for times longer than the occultation period, some of
the consequences of successive illumination periods may be
summed. To avoid this difficulty, Zankel (1971) and others
have resorted to single flash techniques. In this case, the
sample is given a brief (3 us) saturating flash of actinic

light. In some cases it has been necessary to synchronize

12

the flash with a shutter to cut off the tail of the flash
(Zankel, 1971) but in recent years, lasers have been used to
give nanosecond-long pulses (Jursinic and Govindjee, 1977;
Van Best and Duysens, 1975). To increase the signal-to—
noise ratios, flow systems have been used with the flash
technique so that repetitive measurements can be made but
the same biological material is never subjected to more than
one light flash.

Delayed fluorescence can also be triggered by pertur—
bations of the system other than a flash of light. Salt
injections, acid injections, and base injections can all be
done by stopped-flow - a technique in which the two liquid
volumes to be mixed are expelled simultaneously by syringes
into a common tube, where mixing rapidly occurs (2 50 ms).
The biological material can also be perturbed by physical
methods - either electric fields or temperature jumps - with
a consequent emission of light.

All of the above techniques have required a laboratory
setting with bulky power supplies, oscilloscopes etc. Re—
cently, Melcarek et a1. (1977) have described the construct-
ion of a portable solid state device that can simultaneously
monitor prompt and delayed fluorescence. The light source
is a light emitting diode and the light detector is a
HAV4000A large area sensor. With this apparatus they have
measured the effects of temperature on the delayed fluores-
cence and prompt fluorescence of the leaves of chilling

sensitive and chilling resistant plants (Melcarek and

13

Brown, 1977).

Effects of Uncouplers and the Importance of

 

the High Energy State

 

Uncouplers have been known for many years to affect
delayed fluorescence (Mayne, 1968). The bulk of the data
accumulated about these effects have been acquired by workers
using the phosphoroscope technique and have measured the
effect of uncouplers on millisecond-delayed fluorescence.
Almost all uncouplers inhibit such delayed fluorescence.
These data and data to be discussed below have led many in-
vestigators to believe that the so called "high energy state"
is important in the generation of delayed fluorescence from
photosynthetic organisms.

The term "high energy state" is a phrase that was first
used to describe the conditions necessary for photophosphory-
lation by chlorOplasts. The chemiosmotic theory, as formul-
ated by Peter Mitchell (1961, 1966), states that the ender-
gonic phosphorylation of ADP by inorganic phosphate involves
a membrane-bound ATPase that is driven to make ATP by a pH
gradient established across the membrane. This pH gradient
is thought to be generated in plants by the light-driven
oxidation of water and plastohydroquinone on the inside of
the membrane (each reaction producing internal H+). Recently
it has become clear that a membrane potential due to the

unequal distribution of cations and anions can also contribute

l4

much of the energy required for ATP synthesis (Schuldiner
et al., 1973; Graan, 1979).

Uncouplers of photophosphorylation are agents which
abolish the phosphorylation capacity of chloroplasts with-
out inhibiting electron transport. This is thought to occur
by the abolishment of the pH gradient by making the membrane
"leaky" to protons (Good, 1977). The ways in which uncouplers
do this need not concern us here but will be dealt with in
the next part of this thesis. Suffice to say that the hydrogen
ion concentrations on the two sides of a thylakoid membrane
are equal in the presence of uncouplers. Agents exist how-
ever which partition into membranes and can act as carriers
of specific small cations. In the presence of such ionphores,

membrane potentials may be abolished (Pressman, 1976).

Crofts-Fleischman Hypothesis

 

In the presence of either uncouplers or ionphores, the
millisecond-delayed fluorescence is diminished (Mayne, 1967).
This observation led to the suggestion that the millisecond-
delayed fluorescence is somehow stimulated by the high energy
state, a suggestion which was supported by the further obser-
vations that chloroplasts, after being incubated in an
acidic medium, emitted delayed fluorescence when the pH of the
surrounding medium was rapidly raised and a transmembrane
pH gradient was generated (Mayne and Clayton, 1965; Mayne,

1968). This delayed fluorescence induced by a pH increase

15

was abolished by uncouplers. Very soon thereafter, Miles
and Jagendorf (1969) found that chlorOplasts emitted delayed
fluorescence when the external medium was exposed to a rapid
increase in salt concentration. Such an event causes a
membrane potential whenever the less permable anion lags
behind the cation in the diffusion of the salt from outside
to inside.

The above considerations induced Crofts to postulate
(as quoted in Fleischman 1971; see also Crofts et al., 1971)
that the pH and other ion gradients can stimulate delayed
fluorescence. His prOposal begins as a modification of
Lavorel's equation (1968) and takes into account the observ-
ations of Arnold and Azzi (1971) on the glow peaks observed
upon the warming in the dark of frozen, preilluminated

chloroplasts:

dN -Ea/kT

L = T J = -¢ 3? = @NFe (1)

where: L is the intensity of the light emission (in quanta)
® is the efficiency with which excited PS II chloro-
phyll fluoresces
J is the rate of the dark generation of excitons
N is the number of trapped electron or holes
F is a frequency factor containing rate constants and
entropy terms

. -E m
The exponential factor (e a/k-) is the fraction of the

electrons that have enough energy to return to the chloro-

phyll singlet.

l6

Ea is the activation energy

k is the Boltzman constant

T is the absolute temperature
Since the electron transfer from the singlet to the acceptor
occurs rapidly and at very low temperatures, there is prob—
ably no activation energy and Ba may be the difference in
energy between an electron in the acceptor and one in the
chlorOphyll singlet level (Fleischman, 1971).

The initial photochemical charge separation Of photo-
synthesis probably occurs across the membrane, reducing Q
to Q- toward the outside and oxidizing P680 to P680+ toward
the inside (Kraan et al., 1970). A membrane potential
(positive toward the inside) might then facilitate the re-
turn Of an electron from Q- to P680+ and lower the activation
energy required for this process. In considering this effect,
Crofts modified equation (1) to

L = ¢NFe_(Ea - W)/kT

(2)
where W is the membrane potential.

To explain the stimulation Of delayed fluorescence by
a pH gradient, Crofts et al. (1971) suggested that the
electron-carrying redox couples Q/Q- and Z/Z+ are in
equilibrium with pools of secondary donors and acceptors
which are hydrogen-carrying redox couples. These pools are
supposed to be themselves in equilibrium with the aqueous

phases on the Opposite sides of the membrane and their redox

potentials dependent on the pH of these phases. Thus the

17

pH difference across the membrane could increase the
availability of Q- or 2+ (or P680+) or both.

For thermodynamic reasons, Crofts et al. suggested
that the effects of pH and W should be combined into a
single equation using the concept of protonmotive force. As
defined by Mitchell (1966), the protonmotive force (pmf) is
the energetic term which drives phosphorylation and is given
by:

pmf = w — 2.303 gi

ApH.

Thus:

(E - pmf)/kT

L = omFe' a (3)

Kraan et a1 (1970) have proposed a similar model for
the effects of pH gradients on delayed fluorescence. They
assumed however that Q and Z are themselves able to take
up a proton when reduced.

Delayed fluorescence can also be induced by a pH-mediated
reverse electron flow (Shahak et al., 1977). Abrupt changes
in the pH Of the medium are thought to alter the redox
potential of the plastoquinone pool. Reduced molecules of
plastoquinone may then reduce Q to Q— (with a corresponding
increase in the fluorescence yield) in an electron transfer
that is sensitive to DCMU and insensitive to DBMIB.

The Crofts-Fleischman hypothesis has been used extensively
in the past decade to explain the alleged effects Of the high
energy state on delayed fluorescence. Since its promulgation
however, there have been several hints that the hypothesis

might be wrong. Neumann et a1. (1973) and Felker et a1. (1974)

18

showed that under certain conditions, uncoupling by
ammonium salts enhanced millisecond delayed fluorescence
instead of inhibiting it. However, such conditions might

be merely substituting an ammonia gradient for a pH gradient
because it was also shown (Felker et al, 1974) that under
such conditions there is an uptake Of NH4+. Furthermore,
this perplexing stimulation Of the delayed fluorescence

was Observed only with ammonia whereas methylamine, which
seems to uncouple (and be taken up) in the same manner as
ammonia, was inhibitory to millisecond delayed fluorescence.

In this thesis, a laser phosphoroscope capable of
measuring the delayed fluorescence emitted within micro-
seconds has been used to study the effects of uncouplers and
phosphorylating conditions under steady-state conditions.
This thesis also reports the first simultaneous determinations
Of the rate of electron transport (ferricyanide reduction)
and the intensity of microsecond-delayed fluorescence.

In the course Of the studies reported here it was
Observed that uncouplers and phosphorylating conditions
increase rather than decrease the intensity of the delayed
fluorescence when the fluorescence is measured less than
100 microseconds after light extinction. The stimulation
of microsecond delayed fluorescence is correlated with the
rate of electron transport. Nevertheless the often-observed
inhibition of millisecond delayed fluorescence by uncouplers
was confirmed. Thus uncouplers actually increase the

fluorescence which is Observed in the first 100 us but speed

19

the decay in the capacity for delayed fluorescence during
the next 900 us.

Such results will be discussed in the light of the
Crofts—Fleischman model and in the light of other models

that deal with the decay kinetics of delayed fluorescence.

METHODS

Isolation of Chloroplasts

 

Chloroplasts were isolated from commerical spinach
leaves by the methods of Ort and Izawa (1973) or of Hall
et al. (1971). All procedures were carried out in a cold
room at about 4°C and the isolated chloroplast preparations
were stored on ice until used.

In the first method, approximately 30 g Of fresh turgid

spinach leaves (Spinacea Oleracea L.) were washed with cold

 

distilled water and the midribs removed. These leaf sections
were then ground in a blender for 5 s in approximately 90 m1
Of a solution containing 0.3 M NaCl, 0.03 M Tricine-NaOH

(pH 7.8), 3 mM MgC12, and 0.5 mM NazEDTA. After being
filtered through thirty thicknesses of well-rinsed cheese-
cloth, the homogenate was centrifuged for 2 min at 1600 x g.
The chloroplast pellet was resuspended with an artist's
brush in approximately 100 ml of a solution containing

0.2 M sucrose, 5 mM HEPES-NaOH (pH 7.4), 2mM MgClZ, and
centrifuged for 45 s at low speed (<1000 x g). This step
was intended to remove chloroplasts that had clumped together
as well as cellular debris. The green supernatant was then
filtered through two layers Of tissue (Kimwipes) and the
filtrate centrifuged at 1600 x.g for three minutes. The

pellet was washed once and resuspended in a few ml Of the

20

21 '

suspension medium with a final concentration of 1-2 mg
chlorOphyll/ml.

In the second isolation method (Hall et al., 1971),
approximately 50 g of washed spinach leaves were cut into
strips and placed in a blender. Before blending, 100 ml
of a freshly prepared solution of 0.4 M sorbitol, 0.5 M
MES-NaOH (adjusted to pH 6.5), 10 mM NaCl, 5 mM MgC12,
2 mM EDTA, 1% crystalline bovine serum albumin, and 2 mM
ascorbic acid was added. The mixture was then ground for
3 s and filtered through thirty thicknesses Of rinsed
cheesecloth. The filtrate was centrifuged for 90 s at
2900 x g and rapidly slowed. The chloroplast pellet was
resuspended in a few m1 of a solution containing 0.4 mM
sorbitol, 50 mM HEPS (adjusted with NaOH to pH 7.5), 10
mM NaCl, 5 mM MgC12, 1 mM MnCl2 at a concentration of
between 2 and 3 mg chlorophyll/ml.

This second method of chloroplast isolation was used
because other experiments in our lab indicated that chloro-
plasts thus prepared had somewhat greater efficiencies Of
photophosphorylation. In the experiments reported here
however, no noticeable differences were Observed between the
chloroplasts isolated by the two techniques. Therefore most
of the experiments reported here were performed with chloro-
plasts isolated by the first, simple method.

To determine the chloroplyll concentration of the

chlorOplast suspension, a 0.1 ml aliquot of the suspension

22

was diluted with 10 ml of 80% acetone. This was mixed well
and centrifuged at 3000 x g for five minutes. The absorbance
of the resulting supernatant was measured at 645, 663, and
710 nm, with the 710 reading subtracted from the former
values to correct for any "absorbance" not due to chlorophyll
but rather due to some relatively wavelength-independent
scattering. The concentration Of the chlorophyll solution

was calculated with the following formula:

x 8.02) + (A645 x 20.2) = concentration Of
chlorophyll in

ug/ml

(A663

Hydroxylamine Treatment

In one experiment, the electron transport-mediated
oxidation of water was inhibited by pretreating the chloro-
plasts with hydroxylamine and EDTA. Such a treatment is
thought to remove the Mn from the poorly-defined "water—
splitting apparatus" and thus block 02 production (Ort and
Izawa, 1973).

For the treatment, chloroplasts were isolated by the
first method and their concentration adjusted to around
100 ug/ml. A few ml of this suspension were centrifuged at
1600 x g for three minutes and the supernatant removed.

The pellet was resuspended in the NHZOH treatment solution

at a concentration of approximately 100 ug/ml. The NHZOH
treatment solution contained: 0.2 M sucrose, 5 mM HEPES-NaOH

(pH 7.5), 2 mM MgCl 1 mM EDTA, and 3 mM NHZOH. The

2'

23

chloroplasts were incubated in this mixture for twelve
minutes in the dark at room temperature. At the end of

this time, the chloroplast suspension was diluted with the
same cold suspension medium described for the first isolation

procedure, pelleted, and washed twice with the same solution.
Cyanide Treatment

In some experiments it was necessary to vary the rates
of electron transport by pretreating the chlorOplasts for
varying lengths of time with a solution containing KCN
(Ort et al., 1973). This pretreatment was done in an ice
bath in the dark with 0.1 M sucrose, 0.1 M Tricine-NaOH

(pH 8.0), 1 mM MgCl 50 mM KCN, and 50 uM potassium

2:
ferricyanide. These treated chloroplasts were then added
directly into the reaction mixture without removing the
KCN, except by dilution.

Nigericin and gramicidin were added in ethanol. The
final concentration in the reaction mixture was always less

than 3%. This concentration Of ethanol alone had no effect

on the phenomena being studied.

24

Laser’Phosphoroscope

 

The laser phosphoroscope employed was a modification
of the one described by Beall and Haug. Briefly, the light
produced by a continuous output argon laser (principal
lines at 488 and 515 nm) was focused by a microscope
Objective and chOpped at the focal point by a notched disk
rotating at 250 revolutions per 3. This gave a cycle of
1 ms light and 1 ms dark with a light-dark transition time
Of less than 250 ns. The chOpped beam was then collimated
and directed through the bottom of a l x 1 x 4.5 cm quartz
cuvette. Prompt and delayed fluorescence were measured at
a right angle to the actinic beam by a single photomultiplier
tube cooled with a solid C02. A 690 nm interference filter
was placed between the cuvette and the photomultiplier to
shield it from stray actinic light. The output of the
photomultiplier, working into a 509 load, gave discrete
pulses 1-3 ns in length, each pulse associated with individual
photons striking the cathode surface. To measure the delayed
fluorescence, only those pulses generated during a specified
period after light extinction were counted. To do this,
a timing circuit was activated at the instant Of light
extinction. This timing circuit created a counting "window"
by Opening a gate from the photon detector to the counter
for a prescribed time. Under many circumstances this window

Opened 5 Us after light extinction and closed 10 us after

25

light extinction. The pulses arriving during 10,000 of
these gated counting periods were summed (one counting
period every 2 ms for 20 s). The duration of the counting
period and time of Opening of the counting window could be
varied independently so that the rate of decay Of delayed
fluorescence could be determined. When longer delays occured
before the fluorescence was measured, the rate of photon
counting was so much slower that the window was extended
considerably, to 25 us when the delay was 500 us and to
100 us when the delay was 1 ms. However, in the figures,
the data presented have been corrected for this change in
counting time. Prompt fluorescence was measured with the
same photomultiplier by allowing the gate to open to the
counter while the actinic light was still on.

Delayed fluorescence is reported as the ratio of counts
during 5 us divided by the prompt fluorescence in 5 us for
the following reasons: We are interested not so much in the
actual fluorescence as in the thermochemical reactions
responsible for the excitation of the chlorophyll. Since
delayed fluorescence and prompt fluorescence seem to come
from the same chlorophyll (see Introduction) it is reasonable
to assume that the chemically-excited chlorophyll responsible
for delayed fluorescence fluoresces with the same efficiency
as the light-excited chlorophyll responsible for prompt
fluorescence. Thus, dividing by the prompt fluorescence
should help to correct for variations in the fluorescence

yield Of excited chlorOphyll although, to be sure, the prompt

26

fluorescence yield was not measured and could not be
measured at the same instant as the delayed fluorescence.
Nevertheless these corrected values for delayed fluorescence
should reflect the recombination reactions we wish to
measure more accurately than the delayed fluorescence itself.
(However, several assumptions are invoked in these corrections
which are only probable and not certain.) The "corrected"
values have the added advantage of normalizing the results
of the various experiments since each data point represents
a separate experiment and there are inevitable small
variations in the actinic light intensity, chloroplasts
density, and chlorOplast condition.

In the same experiments electron transport was measured
as the reduction of ferricyanide. This was done by Observing
the changes in the ferricyanide absorbance at 420 nm. A
weak light was passed through a 420 nm interference filter
before it passed through the cuvette. This beam was then
detected with a photomultiplier which was protected by
another 420 nm interference filter to screen out the 488 and
515 nm actinic light. The signal from the photomultiplier
was processed by a logarithmic amplifier before it was fed
into a strip chart recorder so that the rates of change in
the concentration of ferricyanide could be directly deter-
mined and recorded.

In all cases the values given for delayed fluorescence
intensity were the mean of 5 determinations. Prompt

fluorescence was measured after 20 s Of preillumination and

27

immediately before and after the delayed fluorescence
determinations. All experiments were conducted at room
temperature, 23°C. The average intensity of the light from
the laser impinging of the reaction cuvette was approximately
80 mW/cm2 (measured with a Yellow Springs Radiometer). The
reaction mixture occupied a cubic volume 1 cm on each side.
The constrast ratio of the measurements of the apparent
delayed fluorescence and actual prompt fluorescence observed
when there was really no delayed fluorescence at all was
determined by replacing the chloroplast-containing reaction
mixture with a solution of chloroplyll in acetone. With
this chlorophyll solution, the counts falsely attributed to
the 5-10 us delayed fluorescence were always less than one
per 10,000 counts Of prompt delayed fluorescence measured
over the same length of time. The intensity of the delayed
fluorescence from chloroplasts was routinely ten times or

more higher than this noise level.

RESULTS

Uncouplers of phosphorylation and phosphorylating con-
ditions (the presence Of ADP + Pi) increase the initial in-
tensity Of delayed fluorescence (Figures 1 and 2). This
stimulation is largest with ammonia (over three-fold at
5 us) and with ammonia persists up to a millisecond after
light extinction. With the other uncouplers and with ADP
+ Pi however, the increases in intensity seen at 5 us are
smaller and the rate of decay of the delayed fluorescene
is larger. Thus at some time between 100 and 500 us after
light extinction the uncouplers and phosphorylating condi-
tions begin to decrease the remaining delayed fluorescence
intensity. It should be noted that these stimulations of
delayed fluorescence are real and not artifacts of the method
of correcting for the fluorescence efficiency of excited
chlorOphyll (see Methods) since they are of a similar
magnitude whether or not such corrections are made. The
results are the same when methylviologen is used as the
electron acceptor for the Hill reaction instead Of ferri-
cyanide. The decay Of the delayed fluorescence does not
follow simple exponential kinetics, as other investigators
have already reported (see Lavorel, 1975a). The log of
the intensity of delayed fluorescence was not a linear
function of the time after light extinction (see Appendix

I, Figures 1 and 2). It is important to note however that
28

Figure 1

29

Delayed fluorescence in spinach chlorOplasts in the
presence and absence of the uncouplers, NH4C1,
nigericin, methylamine-HCl, and gramicidin.

Reaction mixtures contained in 1.0 ml: chloroplasts
containing 5-10 ug chlorophyll; MgClz, 2.0 umol;
Tricine—NaOH (pH 7.8—8.1), 50 umol; potassium
ferricyanide, 0.5 umol; sucrose, 100 umol; gramicidin,
Sug; nigericin, Sug. DF* is 1000 times the ratio

of the number of delayed fluorescence photons
counted in 5 us in the dark to the number of prompt
fluorescence photons counted in 5 us in the light.
At 500 and 1000 us after the light was turned Off,
the periods of counting of delayed fluorescence
photons were increased to 25 and 100 us respectively
with appropriate corrections made to express the
data on the basis of 5 us. Solid lines represent
delayed fluorescence in the presence of the un-
coupler and dotted lines represent delayed fluores—
cence in the absence of the uncoupler.

3O

Am;v Coauocwuxo

Una: umumm TEE.

 

 

 

 

 

£33530

 

 

 

ocflEmamnumz

 

 

 

 

 

 

 

 

Gwowuomflz

oom

 

 

 

 

 

 

the

two

Figure l,

(DF*)
u:

Delayed fluorescence

Figure 2

31

 

 

 

+ADP, +Pi

 

 

 

100 500 1000
Time after light off (Us)

Delayed fluorescence in spinach chloroplasts in
the presence and absence of ADP and orthophosphate.
Reaction conditions and data presentations as in

Figure 1. When added, ADP was 1.5 umol, KZHPO4,
5 umol.

32

the initial downward lepes of the logarithmic decays curves
were nearly equal in the presence or absence of the uncouplers.
It was only at longer times (>100 us) that the rates of

decay in the uncoupled chloroplasts were noticeably greater
than in the controls. Unfortunately the method of measure-
ment did not allow for the collection of enough data points

to detect separate, distinct phases in the decay.

It is clear from these Observations that the delayed
fluorescence cannot be dependent on any uncoupler-inhibited
state of the membrane system. On the contrary, some processes
which are enhanced by uncouplers or by ADP plus Pi must be
responsible for delayed fluorescence. Since all uncouplers
share with ADP + Pi both the ability to increase the electron
transport and the ability to increase 5 us delayed fluore-
scence, a correlation between electron transport rates and
the initial intensity of delayed fluorescence was sought.
Table I of Appendix I confirms this correlation between
electron transport rate and the intensity of 5 us delayed
fluorescence. In these experiments, electron transport was
varied in three ways. (1) It was greatly diminished by
omitting an exogenous electron acceptor. (2) It was almost
abolished with the inhibitor DCMU. (3) Electron transport
was greatly increased by the use of uncouplers in which case,
as already noted, the increase in microsecond-delayed fluore-
scence was marked. In the first two cases with lowered
electron transport rate, the intensity of Us delayed fluore-

scence was much lowered. In the case of DCMU, delayed

33

fluorescence was almost absent. It is particularly important
to note that the increase in delayed fluorescence caused

by uncouplers in the presence Of an exogenous electron
acceptor, did not occur when the electron trasport rate

was limited by the absence of an exogenous electron acceptor.
That is to say uncouplers do not increase the intensity Of
the us-delayed fluorescence when the uncouplers fail to
increase the electron transport rate.

Increases in the concentration of DCMU decreased both
the electron transport rate and the intensity Of the delayed
fluorescence (see Figure 3). The insert of the figure
illustrates the good correlation that exists between these
two parameters. However the disparity between the slopes
of the lines with and without the uncoupler gramicidin
suggest that the uncoupler has some inhibitory effect which
is in addition to its effect on electron transport. A
linear relationship was also found between the rate of
electron transport and the initial delayed fluorescence
when the rate of electron transport was inhibited by cyanide
(Figure 3, Appendix I).

This correlation between the electron transport rate
and the intensity Of the initial delayed fluorescence was
not found when the electron transport was varied in other
ways (i.e. by varying the light intensity, by inhibiting
water oxidation, or by lowering the pH of the medium). In—
creases in light intensity gave increases in delayed fluore-

scence when the electron transport was already light-saturated

Figure 3

34

The effect of the electron transport inhibitor
DCMU on the 5-10 us intensity of delayed fluores-
cence in the presence and absence of gramicidin.
Reaction conditions were the same as in Figure l.
Gramicidin when added was 5 ug. Closed circles
indicate the presence Of gramicidin and Open
circles indicate its absence.

Insert shows the correlation between DF* and the
rate of electron transport under these conditions.
The difference in lepes of the lines with and
without gramicidin shows that gramicidin has some
inhibitory effect on delayed fluorescence in
addition to the effect arising from differences

in electron transport rates.

Rates of electron transport (E.T.) determined
simultaneously as ferricyanide reduction and are
expressed as umol ferricyanide reduced-hr'l-mg
chlorophyll‘l,

(DF*)

5-10 us Delayed Fluorescence

35

 

 

 

 

 

 

 

 

 

 

1c}

20
Concentration of DCMU (nM)

Figure 3.

50

 

36

(see Figure 4 Of Appendix I). This was true whether or not
the chloroplasts had been uncoupled by_gramicidin. Dramatic
increases in "corrected" delayed fluorescence were Observed

in hydroxylamine-treated chloroplasts even though the electron
transport is inhibited by such treatment. Even without the
"correction" which may not be valid, there was little if

any inhibition (see Table I). Finally, at pH 6.5, the large
increases in the rates of electron transport due to uncouplers
were associated with unchanged or decreased intensities of

us delayed fluorescence (see Figure 4).

37

TABLE I
Effects of an Inhibition of Electron Transport on

Delayed Fluorescence when the Inhibition is on the
Oxidizing Side of Photosystem II.*

5-10us 5-1 us
3 DFQ

 

PF (x 10' ) DF‘
experiment 1 —NH20H 163 317 1.94
+NH20H 64 339 5.30
experiment 2 -NH20H 205 436 2.13
+NH20H 86 322 3.74
experiment 3 -NH20H 68 63 0.93
+NH20H 19 64 3.37

 

*

Chloroplast suspensions with a chlorophyll concentration
of 100 ug/ml were treated for twelve minutes in the dark
at room temperature in a solution of 0.2 M sucrose, 5 mM
HEPES-NaOH (pH 7.5), 2 mM MgClz, 1.0 mM EDTA, and 3.0 mM
NHZOH, an irreversible inhibitor of water oxidation. These
chloroplasts were then diluted in the same medium (minus
NHZOH), centrifuged, and the pellet washed twice.

Reaction conditions were the same as Figure 1 except
that methylviologen was substituted for potassium ferri-
cyanide at a concentration of 200 uM in experiments 1 and
2 and at 50 uM in experiment 3. Absolute values of the
directly—measured prompt fluorescence (PF) and delayed
fluorescence (DF) are presented along with the computed
values DF*. The greater inhibition of prompt fluorescence
by the NHZOH treatment was much greater than the inhibitions
Of the 5-10 us delayed fluorescence which suggests that the
thermochemical excitation of the reaction center responsible
for the delayed fluorescence was actually much more frequent
in the presence of the inhibitors.

Figure 4

38

The rate of electron transport and the 5-10 us
delayed fluorescence intensity at pH's from 6.5
to 9.0 in the presence and absence of uncouplers.
Values for the delayed fluorescence intensities
(DF) were corrected for changes in the fluorescence
efficiency as indicated by changes in the prompt
fluorescence (PF) and are plotted in the corrected
form, DF*. Reaction conditions as in Figure l.
Buffers were MOPS (pH 6.5, 7.0), Tricine (pH 7.5,
8.0), and TAPS (pH 8.5, 9.0) - all 50 mM. Solid
lines indicate intensity of 5-10 us DF* and dotted
lines indicate rates of electron transport. Circles
indicate the presence of uncoupler and triangles
indicate its absence.

a) Concentrations Of NH4C1 were varied at each
pH so that the NH3 concentration was constant at
0.355 mM.

b) Concentration of gramicidin was 5 ug/ml.

39

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pH

Figure 4.

DISCUSSION

The initial intensity Of delayed fluorescence

 

In the preceding section, it was demonstrated that
altering the rate Of electron transport can have large
effects on the initial intensity of the delayed fluorescence
from spinach lamellae. The phosphoroscope technique was
employed precisely because it allowed one to measure delayed
fluorescence when the energization Of the membranes and the
redox levels of most Of the electron carriers must have
been close to their steady-state conditions. The dark
period of the phosphoroscope (1 ms) was considerably shorter
than the rate-limiting step of photosynthesis (relaxation
half-time of 10-20 ms, (Stiehl and Witt, 1968; Emerson and
Arnold, 1932). Therefore in the dark, the various pools Of
electron carriers probably were unable to return to their
dark levels. This means that eventually a steady-state
level Of all of the pools Of electron carriers would have
been approached. Those other processes which are connected
to electron transport (membrane potential, ApH etc.) would
presumably also reach a steady-state condition. Of course,
a steady-state of the intermediates close to the photo-
chemistry can never be reached under these conditions
because of the alternating light and dark regime Of the
phosphorOSCOpe. Redox levels of such intermediates at the

end of the dark period may be drastically different from
40

41

those at the beginning of the dark period.

One limitation of the phosphorosc0pe technique that we
have employed is that it is unable to measure the prompt
fluorescence yield at the end of the dark period. It is
thought that since Q is an efficient quencher of fluorescence,
the fluorescence yield is a good indication of the levels of
Q- (Duysens and Sweers, 1963). The corrected delayed fluores-
cence values reported here were on the basis of the fluores-
cence emitted before the light was turned off and so this
correction prOperly applies only for the delayed fluorescence
immediately after light extinction.

Another limitation of this phosphoroscope technique
is that for reliable measurements, each point of any decay
curve required a separate experiment. This made it impossible
to Obtain decay curves with enough data points to identify
separate exponential phases Of the decay (if such separate
phases exist). Such distinct phases have been postulated
on the basis of experiments with single flashes (see Lavorel,
1975b) and their interpretation will be discussed later.

The recombination hypothesis (see Introduction) suggests
that the delayed fluorescence intensity is a function of the
abundance of juxtaposed oxidized primary electron donors and
reduced primary electron acceptors. The only significant
reactions that create these species are light absorption,
and the consequent excitation of the reaction center chloro-
phyll.

Uncouplers are thOught to act directly on the various

42

gradients established across the membrane and indirectly

on the rate of electron transport. Figures 1-4 show that
uncouplers (and other conditions that stimulate the rate

Of electron transport) stimulate the initial intensity of
the delayed fluorescence. Also several conditions which
inhibit electron transport (DCMU, KCN) diminish the

5-10 us delayed fluorescence to a corresponding extent. TO
explain these effects it is necessary to consider the points
of rate-limitation in electron transport and how alterations
in the rates at these points will alter the levels of Q-

and p680+ reached in the light period.

As mentioned above, the rate-limitation of electron
transport comes between the two photosystems (at the point
of the oxidation of plastohydroquinone) which has a relaxa-
tion half-time of around 20 ms (Stiehl and Witt, 1969). The
exact nature of this limitation need not concern us here
but will be dealt with later. Under the conditions employed
here the dark phase of the occulation period (1 ms) was too
short to allow much of the light-reduced plastoquinone to be
reoxidized and it probably stayed mostly reduced. And
since the plastoquinone pool was mostly reduced, it might
be expected that those electron carriers which normally
donate electrons to oxidized plastoquinone (such as Q-) also
stayed mostly reduced. That the levels of Q- were quite
high is born out by measurement Of the prompt fluorescence
yield at the end of the phosphOroscope light cycle.

(Lavorel, 1971).

43

The oxidized P680 is a different matter however. It
must be remembered that electron transport is driven by the
light-driven charge separations that occur at each photo-
system. However no charge separations take place unless the
reaction center is able to accept and use a quantum Of energy
from the light-harvesting complex. This requires that the
reaction center be "open" - a condition in which the primary
donor (P680) is reduced and the primary acceptor (Q) is
oxidized. As mentioned in the previous paragraph however,
under steady-state conditions employed here, the primary
acceptor is likely to be mostly reduced and the reaction
center associated with it therefore closed. To the extent
that electron transport is occurring however, Q-'s are
being reoxidized to Q's and, if the corresponding P680's
are reduced, a charge separation takes place and new P680+
is formed. The reaction rates of the reduction of P680+
are still uncertain. The most reliable data however suggest
that the slowest step in this part of the electron transport
chain is the oxygen—liberating step - a step that has a life-
time of approximately 1 ms (Kok et a1, 1970). This means
that there will certainly be some P680+ at the end of the
light period, the amount of P680+ accumulated being dependent
on the rate of electron transport. Since the levels of P680+
and Q- at the end of the light period presumably determine
the initial levels of delayed fluorescence, stimulations in
electron transport rates might be expected to increase the

initial intensity of the delayed fluorescence. Using similar

44

reasoning, it can be shown that reactions which slow down

the electron transport between the two photosystems - such

as cyanide treatment and additions of DCMU - might be

expected to decrease the initial intensity Of the delayed
fluorescence. Figure 3 of this thesis and Figure 3 of Appendix
I show this to be the case. When electron transport is

limited by either of these inhibitors, there is a linear
relationship between the initial intensity of delayed
fluorescence and the rate of electron transport.

Experiments with NHZOH-treated chloroplasts provide
additional support for a model where the accumulation of
P680+ plays a major role in determining the intensity Of the
delayed fluorescence. Hydroxylamine treatment is thought to
remove the Mn from the "hole collector" involved in the ox-
idation of water, thereby eliminating the donation of electrons
from water to P680+ (Ort and Izawa, 1973). That is to say,
blocking the electron transport on the oxidizing side of
photosystem II should give increased levels of P680+
because the P680+ formed in the light cannot be reduced.
Since the rate of electron transport between the photo-
systems is no longer rate-limiting, the levels of Q_ might
be expected to be lowered somewhat and the decreased levels
of fluorescence tend to support this notion. It is suspected
however, that P680+ is itself an effective quencher of prompt
fluorescence (Mauzerall, 1972; Butler, 1972) so the decreased
levels of prompt fluorescence do not necessarily indicate a

significant decrease in the levels of Q- but may reflect

45

instead the increased levels Of P680+ or may reflect both
factors. It should be mentioned that the quenching by

P680+ can be reasonably expected to affect the light-induced
fluorescence and the chemically induced delayed fluorescence
to a similar extent so that the correction for prompt fluores-
cence which has been used in this thesis is probably still
valid and back-reactions responsible for the delayed fluores-
cence were probably increased by hydroxylamine treatment.

It now seems clear that the initial intensity of delayed
fluorescence is a reflection Of the rates Of electron transfer
to and from the reaction center of photosystem II which in
turn affect the levels of Q_ and P680+ reached in the light
period. It may be however, that these Observed relationships
apply only under a narrow range of conditions. For instance,
lowering the pH from 8.1 to 6.5 in the absence of an uncoupler
lowered the rate of electron transport but increased greatly
the delayed fluorescence (Figure 4). In addition, the
electron transport can be saturated with light but further
increases in light intensity still result in increased
intensities of delayed fluorescence (Figure 4, Appendix I).

It is possible that the delayed fluorescence at
5-10 us may not reflect the relative amounts of P680+ and
Q- at the very instant of light extinction. It is still
even possible that the effects of uncouplers on the 5-10 us
delayed fluorescence represent inhibitions Of some component

of a decay which has a time constant much less than 5 us.

46

Decay;of‘Delayed‘Fluorescence

 

Most delayed fluorescence experiments heretofore
reported can be put into two categories, those using phosphor—
oscope techniques and those using single flash techniques.
Phosphoroscope experiments purport to look at the effect of
"steady-state" conditions on delayed fluorescence - usually
at a significant length of time after light extinction (milli-
seconds). Single flash techniques however look at the other
side of the coin. Any build up of gradients or filling of
electron-carrier pools (such as with closely-spaced flashes)
is usually avoided but the time between the measurement and
light extinction (microseconds) is several orders of magnitude
less than with the phosphoroscope. The flash experiments
attempt to look at the fast recovery kinetics of the reaction
centers in order to establish the properties of the components
associated with photosystem II. These two approaches leave a
wide gap between them however, a gap which the experiments
detailed here attempt to bridge. The laser phosphoroscope
allows the measurement Of fast decay kinetics under steady-
state conditions. Such determinations are also possible with
steady preillumination before a flash or with a rapid series
of flashes and such an approach has recently been attempted
(Jursinic et al., 1978). The results of these experiments
will be discussed further.

In the preceding section we discussed the effect of

uncouplers on the initial (5 us) intensity of delayed

47

fluorescence and presented a model which attempted to

explain these effects on the basis of rates Of electron
transport determining the levels of P680+ and Q- formed

in the light. In this section the electron transport-

centered model is extended to explain the effects Of uncouplers
on the rate of decay of the intensity of the delayed fluore-
scence.

The major points to explain are:

a) Uncouplers have no effect on the rate of decay of
the delayed fluorescence measured up to 100 us after light
extinction.

b) At times after light extinction greater than 100 us,
the rate of the decay of delayed fluorescence in the presence
of uncouplers is greater than the rate of decay in the absence
of uncouplers.

Before attempting to explain these Observations, it is
necessary to discuss briefly the meaning of a decay curve for
delayed fluorescence.

As mentioned above, the intensity of delayed fluorescence
at any time is thought to be a function of the number of
reaction centers at any instant that have both a reduced
primary electron acceptor (Q-) and an oxidized reaction
center chlorOphyll (P680+). Delayed fluorescence thus is
considered to be a measure of the back-reaction of Q- and
P680+. If there are no reactions that compete with these
back-reactions, the decay of delayed fluorescence would

probably follow simple exponential kinetics, and a plot of

48

the log of the delayed fluorescence intensity versus time
after light extinction would give a straight line. One
observes however that the decay of delayed fluorescence is
not a simple exponential but is more complex. An initially
rapid rate of decay gives way to a slower rate of decay.
However separate decay phases are not always easy to dis-
tinguish (see Figure 1, Appendix I). Deviations from simple
exponential may be due to reactions Of Q- and P680+ other
then the light-producing back reaction. The reduction of
P680+ by electrons from water and the oxidation of Q-
by plastoquinone and ultimately by photosystem I are such
reactions.

Detailed analyses Of the various phases Of the decay
of the delayed fluorescence observed after saturating flashes
of light have appeared quite Often in the literature (Lavorel
1975b; Zankel. 1971) and are perhaps a good beginning point
for this section Of the discussion. However the conditions
most Often employed for these studies (single saturating
flashes with dark-adapted chloroplasts or algae) are
significantly different from those used here (phosphoroscope
and steady-state electron transport conditions) so that the
observations obtained in the earlier studies may apply in
only ageneral way to the results reported here.

It is well known that the rate of reduction Of P680+
in the dark is significantly faster than the oxidation Of
Q“. The values of these reactions are commonly Obtained by

such physical mean as rapid light and EPR spectroscopy, prompt

49

fluorescence, and 02 yield.

P680+ is rapidly reduced by the first secondary donor
(Z) and the half-time for this reaction is probably around
30 ns (VanBest and Mathis, 1978). In fact, these two electron
carriers may be in direct equilibrium. Z+ is reduced by
the "hole collector", with rates of reduction determined by
the "S-state" of the hole collector. Half times for this
reaction range from 100 us (SO, 81) to 1 ms (53) (Babcock
et al., 1976). This means that if one was to look at the
reduction of P680+, it would have an initial fast phase of
reduction which reflects the donation of electrons from
those Z's that are reduced. 'There would also be slower
phases that reflect the reduction of P680+ by Z's that
were initially oxidized and reduce P680+ after having
accepted an electron from the hole collector.

On the Q side: The reoxidation of Q- is reported to
have a half-time of about 0.6 ms (Forbush and Kok, 1968;
Stiehl and Witt, 1969) whereas the reoxidation of half of
the plastoquinone pool takes a much longer time, about
20 ms (Stiehl and Witt, 1968). The latter reaction is
sensitive to uncouplers and Rumberg and Siggel (1969)
report that the half-time value decreases to about 6 ms in
the presence of BUM gramicidin at pH 8.0. It should be
emphasized that pool sizes are important when considering
the oxidation 0f Q- by plastoquinone. On the basis of these
data (Rumberg and Siggel 1969), it can be calculated that one

millisecond after light extinction, approximately 1% Of the

50

plastoquinone pool would have been reoxidized in the absence
Of gramicidin and over 10% would have been reoxidized in the
presence of gramicidin. Estimates vary on the number of
active plastoquinone molecules per Q molecule but even with
a ratio of 5 to l (which means 10 electron equivalents per
1) at the end of one millisecond, the Q- might be expected
to be about 10% reoxidized in the absence of gramicidin and
70% reoxidized in the presence of gramicidin.

TO explain the observations of the effects of uncouplers
on the rates of decay Of delayed fluorescence it is necessary
to remember that uncouplers increase the rate of oxidation of
plastoquinone. Therefore it seems probable that the intensity
Of delayed fluorescence is determined by the amount of P680+
during the first 100 us, and increases in the rate Of the
oxidation of plastoquinone should have no effect on this
initial decay. At longer times however, the amount of Q—
has decayed to a point where it determines the rate of the
back-reactions with the rare remaining P680+ centers. Under
these conditions, uncouplers which increase the rate of
decay Of Q- lower the intensity Of the delayed fluorescence.

It seems therefore possible to explain the inhibition
of the delayed fluorescence emitted at milliseconds after
light off on the basis of their effects on electron transport
rather than their effects on ion gradients.

The model presented here implies that membrane potentials
have no appreciable effect on the microsecond delayed fluores-

cence. This corroborates the observation of Jursinic et a1.

51

(1978) that uncouplers had no effect on the microsecond
delayed fluorescence Observed after a single flash with no
preillumination.

In this section Of the thesis a model has been pre-
sented which attempts to explain the effects of uncouplers
on delayed fluorescence on the basis of their effects on the
rate of electron transport between the two photosystems. It
should be emphasized however that the quantum efficiency Of

delayed fluorescence is extremely small (10-4

). It may be
that all of delayed fluorescence represents aberrant processes

which have little to do with normal photosynthesis.

SECTION II
THE EFFECTS OF AMINES AND OTHER UNCOUPLERS ON

ELECTRON TRANSPORT, ATP SYNTHESIS, AND P/E2

INTRODUCTION

This section Of the thesis deals with uncoupler
effects that are probably more direct than their effects on
delayed fluorescence - namely their effects on the rates of
electron transport, the rates Of phosphorylation and on the
magnitude of pH gradients. The literature pertaining to
uncouplers is quite extensive and has been periodically re-
viewed (Good et al., 1966; Gomez-Payou and Gomez-Lujero,
1977; Good, 1977). This section will present the basic
phenomena attributed to uncouplers and briefly discuss the
accumulating data which suggest that unified theories Of
amine uncoupling should be reconsidered.

It is impossible to discuss uncouplers in the absence
of a discussion of photophosphorylation. The coupling
intimated by the term "uncoupler" is between electron trans-
port and the synthesis of ATP. The widely accepted chemi-
osmotic theory (Mitchell, 1966) states that these two
processes are both intimately involved with a pH gradient
across the lamellar membrane and, indeed, electron trans-
port generates a pH gradient and ATP synthesis can be in-
duced by a pH gradient. The evidence for these statements

will be briefly discussed.

52

53

Electron Transport Generates ang Gradient

 

The primary evidence is:

a) If chlorOplasts are allowed to perform either
noncyclic or cyclic electron transport in a weakly-buffered
solution, there is a light—dependent rise of the pH of the
external medium (Neumann and Jagendorf, 1964).

b) Monofunctional amines with high pK's are taken up
by chlorOplasts during electron transport. This has been
shown either by monitoring the disappearance Of a radioactive
amine from the external medium or by measuring the internal
concentration Of a radioactive amine after rapidly centrifuging
the chloroplasts through a layer Of silicone (Portis and
McCarty, 1976). The uptake Of the amines is thought to be
a result of the rapid equilibration across the membrane of the
lipid-soluble free base and the unequal distributions Of the
charged and uncharged amines due to the different pH's of the
separate sides Of the membrane. Assuming ready equilibrium
Of the uncharged amine, it can easily be shown that
[H+]in/[H+]out = [charged aminelin/[charged amine]out.

c) A pH-indicating dye (neutral red) can be allowed
to enter the internal space of the chlorOplast lamellae and
when pH changes in the external medium are prevented by
buffering, changes in the dye absorbance are presumed to
indicate changes in the pH of the internal space (Auslander
and Junge, 1975). This approach is very speculative however

because Of problems of binding of the dye and the resultant

54

change Of its pK.

The above observations led to the suggestion that
both noncyclic and cyclic electron transport involve an
electron carrier which accepts a proton when reduced on
the outside of the membrane and donates a proton when ox-
idized on the inside of the membrane. One such electron
and proton carrier is presumed to be a plastoquinone
because:

a) The number of active plastoquinone molecules is
greater than those of any other member of the electron trans-
port chain and this pool may be large enough to span the
membrane. It is also possible that the plastoquinone which
is lipid-soluble may be able to diffuse across the membrane
(Witt, 1979).

b) The kinetics of internal H+ release parallel the
kinetics Of the oxidation of plastohydroquinone over a range
of 20-fold variation (Tiemann et al., 1979).

c) The number of protons taken up from the external
phase increases in parallel with the number of electrons

taken up by the plastoquinone pool (Tiemann et al., 1979).

Ang Gradient Generates ATP

 

Much data has been accumulated that suppports the
hypothesis that a pH gradient can drive ATP synthesis (see
Jagendorf, 1977). ChlorOplasts synthesize ATP from ADP and

Pi when a pH gradient is imposed across their membranes. The

55

gradient can be formed by incubating the chloroplasts at a
low pH and then rapidly raising the pH of the external
medium (Jagendorf and Uribe, 1966). This acid-base induced
ATP synthesis is sensitive to internal buffering, weak
amines which store protons giving greater yields Of ATP.

Another indication of the connection between the pH and
ATP synthesis is the Observation that under suitable
conditions, ATPase, will hydrolyze ATP with a concomitant
"pumping" of protons into the internal aqueous space (Crofts,
1966).

Experiments in several laboratories have shown, however,
that whereas a proton gradient of 2 to 3 units is sufficient
to drive ATP synthesis, such a large pH gradient is not
always necessary and that a membrane potential can contribute
much of the energy required for ATP synthesis (Schuldiner

et al., 1973; Graan, 1979).

Uncoupling and the Control Of

 

the Rate of Electron Transport

 

The rate of noncylic electron transport varies with
pH and is greatest between pH 8.0 and 8.5 in the absence of
uncouplers. This maximum shifts to around pH 6.5 in the
presence of uncouplers (Good et al., 1966). It is well
known that a main rate-limiting step in electron transport
is located between the two photosystems and probably reflects

the rate of oxidation of plastohydroquinone. It would not

56

be surprizing if the step were sensitive to pH because the
oxidation of PQH2 releases protons, and a high concentration
of H+ (low pH) might exert a back-pressure on this reaction.
Some investigators have found that the controlling factor is
the internal pH (Rumberg and Siggel, 1969; Siggel, 1974).
However their conclusions were based on the assumption that
pH gradients are completely abolished by 3 uM gramicidin,

an assumption that was not subsequently supported by the

pH determinations of Portis and McCarty (1976). Bamberger
et a1. (1973) have suggested that the controlling value is
instead the average Of the internal and external pH's.
However as was shown by Ort (1976) with dark-adapted chloro-
plasts, the initial rate of electron transport is rapid but
slows to steady-state rates within 50 ms. The addition of

a permeant buffer which presumably prevents any rapid de-
crease in the pH of the internal space, does not significantly
delay the time at which this slowdown occurs. Thus the pH
Of the inner aqueous phase may have nothing to do with the
primary control of electron transport.

A general definition Of uncoupling is any treatment
which inhibits phosphorylation without causing corresponding
inhibitions in electron transport, or increases electron
transport without causing a corresponding increase in
phosphorylation (Good, 1977). However such a definition is
not completely satisfactory because the omission Of ADP or
Pi or the addition of an energy transfer inhibitor, by in-

hibiting phosphorylation would act as "uncoupling" of

57

chloroplasts performing phosphorylation at site II only,
since the rate of electron transport at this site is
usually independent of phosphorylation (Gould and Izawa, 1973).
Uncoupling is therefore better defined as the destruction
of the energized state of the lamellar membrane (Good, 1977).
If, in fact, electrochemical gradients are required for
phosphorylation, then uncouplers are agents which make the
membrane "leaky" to protons and other ions.

The uncoupling effects of coupling factor removal
(which forms nonspecific holes in the membrane- Avron, 1963)
and phosphate analogs (which replace P1 to form unstable
intermediates with ADP- Avron and Jagendorf, 1959; Avron
and Shavit, 1965) are more malfunctions of the coupling
factor than of the membrane per se and will not be considered

here.

”Uncouplings‘by'Amines

 

In 1959, Krogmann et al. reported that ammonium salts
can act as uncouplers. It is now known that many amines with
membrane-permeating unprotonated (hence uncharged) forms can
abolish pH gradients across the lamellar membranes of
illuminated chlorOplasts (Good, 1960; Hind and Whittingham,
1963). The acidification of the inner space which would be
anticipated in the absence of an amine is thought to cause
a decrease in the internal concentration of the free base

of the amine. Since the internal concentration of the free

58

base has been lowered, a gradient is established and more of
the free base diffuses across the lamellar membrane and into
the inner space. However, this uptake does not by itself
abolish the acidification of the internal space. Since an
uptake of the uncharged form of the amine fails to counteract
the uptake of positive charges (proton), the internal space
becomes positively charged; this charge may be sufficient

to drive the charged protonated form of the amine back out

of the chloroplast and it is this influx of unprotonated
amine and efflux of protonated amine which effectively
abolishes the acidification of the interior. In the presence
of permeant aniOns, the uptake of the uncharged amine is
accompanied by an uptake Of an anion and there may be no
increase in the net positive charge Of the internal space
sufficient to expel the protonated amine. Thus in the
presence of permeant anions, amines cause a swelling of the
lamellae but often fail to prevent the acidification of the

interior, and therefore uncouple less effectively.

Uncoupling by Weak Acids

 

Uncoupling can be accomplished by weak acids if their
anions are relatively lipid soluble. In this case there is
no uptake Of the uncoupler but rather the uncoupler shuttles
protons across the membrane. Carbonylcyanide phenylhydrazones
(e.g. CCCP and FCCP) are perhaps the best examples of these
since they uncouple effectively at low concentrations.

Indophenols at lower pH's which allow the formation of some

59

of the red, unprotonated form, also seem to uncouple in this
manner but they are not nearly as effective. They are more
commonly used as electrons acceptors which only incidentally

uncouple and then only partially, depending on pH.
Uncoupling‘by‘ionophores

IonOphores are a group of lipid-soluble molecules with
backbones of diverse structures that contain strategically-
placed oxygens. The conformation of the molecules are such
that the oxygens form a central ring or cavity in which small
cations may become sequestered (Pressman, 1976). This
sequestering effectively buries the charge of the cation
within the ionOphore molecule with the result that they can
often carry ions while at the same time being lipid soluble.
The structure of the ionOphore imparts an element of selec-
tivity in the cations that can be transported. Those
ionOphOres of greatest interest to uncoupling can transport
protons and other small univalent cations. Nigericin cata-

lyzes an exchange of H+ for K+ across the membrane.

Uncoupling'by‘Quasiionophores

 

Though often considered along with ionophores just
mentioned,gramicidin is not an ionOphore in the stricktest
sense. It forms ion-conducting channels across the full

thickness of membranes (Pressman, 1976). These pores are

60

also very specific for inorganic monovalent cations, and,
since the action of gramicidin does not depend on its
diffusion across the membrane, uncoupling can be very

effective.

Unconventional Uncoupling

 

Many other substances can uncouple chloroplasts and
their mechanisms are Often not well understood (for a
review, see Good, 1977). The most interesting of these is
atebrin, an aliphatic amine uncoupler containing an aromatic
ring. It clearly has some mechanism distinct from that of
simple amines for several reasons: a) Uncoupling by
atebrin is accompanied by a shrinking of the chloroplasts
instead of a swelling; and b) Atebrin is reputed to uncouple
even while bound to large sepharose beads (Kraayenhof and
Slayter, 1975). This second effect has also been found with
long alkylamines (n>12) bound to sepharose (Lotina et al.,
1979).

There are striking differences among the effects of
uncouplers and, although many abolish gradients by increasing
the rates of ion diffusion, different types of "leaks" must
be involved. Some uncouplers interact in ways which cannot
yet be explained.

There seems to be a hierarchy of shrinking and swelling
effects: carbonylcyanide phenylhydrazones abolish all volume
changes whether or not amines or atebrin are present. Amines

cause swelling whether or not atebrin is present. Also the

61

removal of coupling factor in no way interferes with the
shrinking or swelling induced by atebrin or amines although
chloroplasts uncoupled by removal of coupling factor alone
neither shrink nor swell.

Chloroplasts have different sensitivities to uncouplers
under different conditions and changes in conditions may not
change these sensitivities in parallel. Under steady-state
conditions, low concentrations Of CCCP inhibit phosphorylation
quite effectively at low light intensities while at higher
light intensities the same concentrations have little effect
(Saha et al., 1970). Octylamine and FCCP inhibit ATP synthesis
with short flashes of light at concentrations that hardly
effect steady-state phosphorylation (Ort, 1978) and acid-base
phosphorylation driven by small pH changes is much more
sensitive to proton-carrying uncouplers than is steady-state
phosphorylation (Graan, 1979).

These intriguing differences were a stimulus for the
studies reported here on the effects Of uncouplers on electron
transport and photOphosphorylation. Since there is a dearth
of reports in the literature on surveys of the effects of
uncouplers at different light intensities and pH's etc.,

such an investigation was though advisable as a starting point.

METHODS

Isolation of chloroplasts was as described in the
Methods section Of Section I.

Noncyclic electron transport was measured as the re-
duction of ferricyanide and was measured as the decrease in
absorbance at 420 nm. Illumination was provided by the
500 watt bulb of a slide projector the light of which was
filtered by a dilute copper sulfate solution (to screen
out infrared radiation) and a red glass filter. Temperature
was maintained at 18°C with circulating water from a thermo-
statted water bath through a solid brass cuvette holder.

The red actinic light was kept out Of the 420 nm measuring
device by the use of a complementary blue filter.

ATP synthesis was measured as the radioactivity
remaining after the extraction with organic solvents of
the unreacted 32Pi as phosphomolybic acid. This was done
by removing a 1.0 ml aliquot of the mixture and mixing it
with 9.0 ml of cold 10% perchloric acid saturated with
hexanol in a 20 mm x 150 mm pyrex test tube. TO this was
added 1.0 ml Of 20% (w/v) solution of ammonium molybdate
and the solution was stirred well for 60 s with a glass
plunger. After five min, 16 m1 Of hexanol (saturated with
10% perchloric acid) were added and stirred with a glass
plunger. After the two layers had separated the top (organic)

layer was removed by suction and the lower layer was subjected
62

63

to gravity filtration through pre-wetted filter paper.

This filtration step removed particulate matter and any
remaining minute drops of the organic (and highly radio-
active) layer. To the filtrate was added 0.1 ml more of the
ammonium molybdate solution and another 16 ml of the hexanol
solution. After 60 s of vigorous mixing, 0.1 m1 of 0.1 M

Na HPO

2 4
the removal of the remaining ammonium molybdate. After

was added to the test tube in order to facilitate

5 min, the two layers were again mixed, the phases allowed
to separate, and the top layer removed with suction. If
the bottom solution showed any traces of a yellow color, it
was once more extracted with hexanol and the tOp layer
removed.

Radioactivity in the final aqueous phase was determined
by counting the scintillations due to Cerenkov radiation
and counts associated with ATP were made by comparisons with

accurate dilutions of the 32Pi stock.

RESULTS AND DISCUSSION

As a starting point for these studies of the effects
of uncouplers, a series of experiments were conducted to
determine the effective concentrations at pH's from 6.5 to
9.0. In these studies, basal (non—phosphorylating) and
coupled electron transport, ATP synthesis, and phosphorylation
efficiency (P/ez) were determined (Figure 5).

The major observations were :

a) As has long been known, basal and coupled electron
transport in the absence Of uncouplers have their maximum
rates between pH 8.0 and 8.5. P/e2 has a broad maximum
between 8.0 and 9.0.

b) The highest rates of electron transport were Obtained
at pH 6.5 when the concentrations of the uncouplers were
high enough to give maximum rates at the pH investigated.

c) All uncouplers gave their largest stimulations of
electron transport at the lower pH's. This is, in part,
because of basal electron transport is then so slow.

d) At pH's well above their pK's, amines did not
uncouple even though they were effective uncouplers at or
below their pK's.

e) In the presence of some amines and at some pH's large
increases in electron transport were observed with little or

no decrease in ATP synthesis.

64

Figure 5

65

Effects of uncouplers on electron transport and
photophosphorylation at pH's from 6.5 to 9.0.
Reaction mixtures contained in 2.0 ml: chloroplasts
with 30 ug chlorophyll; sorbitol, 0.4 umol; MgClz,
4.0 umol; potassium chloride, 20 umol; potggsium
ferricyanide, 1.0 umol; ADP, 1.5 umol; K2H P04,

10 umol. Buffers used were MOPS-NaOH (pH 6.5,
7.0), Tricine-NaOH (pH 7.5, 8.0), and TAPS (pH 8.5,
9.0) - all 100 umol.

For determinations Of electron transport in the
absence of phosphorylation (Open circles), the
K2H32po4 and the ADP were omitted (a,b,c,d) or
both the K2H32PO4 and the ADP were omitted (e,f,

glhlilj)° P/e2 (closed circles) represents the
phosphorylating efficiency of the electron transport;
the number Of molecules Of ATP formed for each

pair of electrons transferred from water to
ferricyanide. The phosphorylation rates (as umoles
ATP formed/hromg Chl) are indicated by closed
triangles and the rates of electron transport when
phosphorylation is occurring (as umoles ferricyanide
reduced/hr-mg Chl) are indicated by Open triangles.

66

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76

Points a), b) and c) are commonly observed and perhaps
need no elaboration beyond the explanation given in the
introduction of this section.

Point d) is an indication that amines with low pK's
behave differently from those with higher pK's. The few
amines mentioned have pK's around 7.0. It has long been
thought that weak amines such as those listed are poor un-
couplers. If one limits one's observation to high pH's only,
this is true. However at lower pH's, large increases in
electron transport are possible at concentrations Of amine
that are not unreasonable. These differences in effectiveness
at high and low pH are easily explained however (see
Schuldiner et al., 1972). It should be remembered that the
uncoupling by amines is due to the different pH's on the
two sides of the membrane. This difference in pH normally
results in differences in the concentrations of the uncharged
form Of either side of the membrane and diffusion of this
form across the membrane followed by the extrusion Of the
protonated amine neutralizes the internal pH. At pH's well
above the pK of the amine however, the amine is almost
entirely in the free base form and dr0ps in the internal
pH are not sufficient to lower the internal concentration
Of the free base sufficiently to cause a massive uptake and
release of the amine which seems required for effective
uncoupling.

Point e). The fact that conditions which increase the

rate of non-phosphorylating electron transport (i.e. uncoupling)

77

Often do not significantly inhibit phosphorylation and can
sometimes actually increase the rate of ATP synthesis seems
inconsistant with the very concept of coupling. The failure
of certain amounts of uncoupled electron transport to de—
crease phosphorylation can be understood if the actual coupled
transport reaction is strongly rate-limiting; that is to
say, if all other reactions are in excess. Then the coupled
reaction might be in part uncoupled or become non-phosphoryl-
ating without diminishing the supply Of electrons for the
coupled reaction. In this context it should be remembered
that the phenomenon we are discussing is most pronounced at
low pH's where those electron transport reactions associated
with phosphorylation are indeed very slow and probably rate-
limiting. However this interpretation does not explain the
actual stimulations of phosphorylation observed.

There have been some other reports Of small (< 20%)
increases in the rate Of electron transport with no decrease
in phosphorylation with low concentrations of methylamine
(see Good at al., 1966) but no reports with the large
discrepancies reported here. In fact, at pH 6.5, "uncoupling"
by HEM at 2.5 mM gave a 1.85-fold increase in the rate of
ATP synthesis with a 3-fold stimulation of electron transport!

Other amines with pK's near 7.0 seem to behave in a
similar if not identical manner (see Table II). The weaker
amines give much less Of a decrease in the rate Of ATP
synthesis when the electron transport rate is increased 4-fold.

The differences between the amines with pK's near 7.0 might

78

TABLE II. Correlations Between the Stimulation of Electron
Transport and the Inhibition of Phosphorylation
by Uncouplers at pH 6.5*.

Increase in %Inhibition of

 

Uncoupler pK E.T. rate Phosphorylation
Octylamine 10.6 4-fold 100
Gramicidin - 4 " 100
CCCP - 4 " 100
NH4C1 9.25 4 " 82
Methylamine 10.6 4 " 65
Imidazole 7.0 4 " 52
2,4—lutidine 6.7 4 " 43
2,6-lutidine 6.7 4 " 33
DMAP 7.0 4 " 22
HEM 7.0 4 " 0

 

* Data were calculated from the results presented in Figure 5

79

be thought to be due tO differences in the lipid-solubility
of their uncharged forms. However HEM and imidazole, which
gave large differences in the lack of inhibition of ATP
synthesis, have the same pK and partition to the same
extent between water and petroleum ether (data not shown).

In order to distinguish further the effects Of amines
with low pK's and high pK's, the effect of light intensity
was investigated at pH 7.5 (Figure 6). Surprisingly, light
intensity is an important factor in the stimulation of
steady-state phosphorylation by amines and not simply through
any role such as rate limitation. Even methylamine, which
diminished phosphorylation at high light intensities (100%
relative units), can stimulate phosphorylation at lower
light intensities (16—43% relative units). Ammonia however
decreased the rate of ATP synthesis at all light intensities.

The reasons for these stimulations of phosphorylation
by amines are Obscure and speculations at this point may be
premature. It is clear however that uncoupling by amines is
not as straightforward as once thought and it is possible to
get large differences between uncoupling as determined by
increases in electron transport and as determined by an
inhibition of ATP synthesis.

Since amines with pK's near 7.0 clearly need more
investigation, many of them were checked for their ability
to stimulate electron transport at pH 6.5. The results of
these experiments are presented in Table III. All of the

amines tested could uncouple to some extent. However the

Figure 6

80

The effects of light intensity on the uncoupling
by amines. Reaction conditions in 2.0 ml: chloro-
plast suspension containing 30 ug chlorophyll;
sorbitol, 0.2 M; Mgc12, 2.0 mM; Na2H32PO4, 10 mM;
potassium chloride, 10 mM, potassium ferricyanide,
0.5 mM; ADP, 0.75 mM; Tricine—NaOH (pH 7.5),

50 mM.

Arrows indicate the rates of electron transport and
the bars indicate the rates of phosphorylation at
the millimolar concentrations shown. Maximum

light intensity {100 %) was approximately

400 Kerg-cm‘ os' .

(umoles ferricyanide reduced/hr-mg Chl)

Rate of Electron Transport

1000

800 »

600

400

200

1000

800

600

400

200 ,

500 ’

400

300 '

200

100

81

 

:4
l
l
l
a:
ll

 

HEM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0 mM
5 mM

+fl. c = 20 mM

'150

100

50

 

 

size size alac al)c a1)c alac

 

NH C1

1 f
'1 T b

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0 mM
1 mM
5 mM

1

flOO

50

 

 

in .. c
(11144];

abc abc abc ab abc abc

 

 

 

:f
ll

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

a be aibc alac alac alac atac
100% 43% 33% 16% 6.8% 5.3%

Relative Light Intensity

Figure 6

F r‘ Methylamine
F 1. a = 0 mM
, F
#1” - b=1mM
5 mM

 

‘100

1 [EL

50

(umoles ATP formed/hr-mg Chl)

Rate of ATP Synthesis

82

TABLE III. A Survey of the Effectiveness of Amines with pK's
near Neutrality in Stimulating Electron Transport

 

at pH 6.5*.
Electron Trans-

AMINE pK port Stimulation
2,4,6-collidine 7.5 11-fold
N-methylmorpholine 7.4 26 "
l-ethylimidazole 7.3 18 "
N-allylmorpholine 7.05 23 "
N—Bhydroxyethylmorpholine 7.0 21 ”"
imidazole 7.0 22 "
3-dimethylaminopropionitri1e 7.0 15 "
bis(Z-chloroethyl)amine 7.0 9 "
1-dimethylamino-2-propyne 7.0 22 "
2-aminOpyridine 6.8 16 "
2,4-lutidine 6.7 21 "
2,6—lutidine 6.7 12 "
S-ethyl—Z—methylpyridine 6.5 3 "
3,4-lutidine 6.5 8 "
methylaminoacetonitrile 5.2 5 "

 

* O 0 I
Reaction conditions as in Figure 5. Values given for stim-
ulation are a ratio Of the maximum uncoupled rate of electron

transport to the basal rate of electron transport.

83

substituted pyridines tended to give progressive inhibitions

and should be used with caution.

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APPENDICES

APPENDIX I
STIMULATION OF MICROSECOND-DELAYED FLUORESCENCE
FROM SPINACH CHLOROPLASTS BY UNCOUPLERS

AND BY PHOSPHORYLATION

94

446

Biochimica et Biophysica Acta, 504 (1978) 446—455
© Elsevier/North-Holland Biomedical Press

BBA 47580

STIMULATION OF MICROSECOND-DELAYED FLUORESCENCE FROM
SPINACH CHLOROPLASTS BY UNCOUPLERS AND BY
PHOSPHORYLATION

DUNCAN H. BELL a, ALFRED HAUG b and NORMAN E. GOOD “

3 Dept. of Botany and Plant Pathology, b MS U/ERDA Plant Research Laboratory, Michigan
State University, East Lansing, Mich. 48824 (U.S.A.)

(Received May 2nd, 1978)

Summary

Delayed fluorescence, as measured with a laser phosphoroscope, is stimu-
lated not inhibited by uncouplers during the first 100 as after the light is
turned off. This is true only when uncouplers cause an increase in the rate of
electron transport. When ADP and P, cause an increase in the electron transport
rate, microsecond-delayed fluorescence is also increased. Indeed, there is a com-
plex quantitative relationship between the rate of electron transport and the
initial intensity of delayed fluorescence under a wide range of conditions.

Uncouplers or ADP and P, also increase the rate of decay of delayed fluores-
cence so that after about 150 as they become inhibitory, as already reported
by many authors.

Microsecond-delayed fluorescence continues to rise with rising light.inten-
sities long after the rate of reduction of exogenous acceptor is light-saturated.

These observations suggest a correlation of the rate of electron transport
both with the intensity of the 5—100 its-delayed fluorescence and with the
rate of decay in the intensity of delayed fluorescence. The data imply that the
decrease in intensity of millisecond-delayed fluorescence which has often been
noted with uncouplers is probably not due to the elimination of a membrane
potential. It seems more likely that the decrease in millisecond-delayed fluor-
escence is a reflection of the rate of disappearance of some other electron
transport-generated condition, a condition which is uncoupler-insensitive. Cer-
tainly stimulations of microsecond-delayed fluorescence by electron transport
which has been uncoupled by gramicidin suggest that ion gradients are not an
essential component of the conditions responsible for delayed fluorescence.

 

 

Abbreviations: DCMU. 3-(3.4-dichlorophenyl)-l.l-dimethylures; HEPES.N-2-hydroxyethylpiperazine-N'-
ethanesulfonic acid; Tricine. N-tris(hydroxymethyl)methyl¢lycine. DF“. values of delayed fluorescence
corrected for changes in the efficiency of fluorescence of excited chlorophyll and expressed as the ratio
of delayed fluorescence counts to prompt fluorescence counts multiplied by 1000.

95

447
Introduction

The delayed fluorescence of green cells and isolated chloroplasts appears to
be a reversal of the initial photochemical and thermochemical events asso"-'
ciated with Photosystem II [1] (for a review, see ref. 2). This fluorescence per-
sists for milliseconds or longer and is envisioned as arising from a charge recomv
bination within the Photosystem II reaction center involving the reduced prim-
ary electron acceptor (Q') and the oxidized form of some primary electron
donor [3].

Uncouplers of photophosphorylation inhibit that part of the fluorescence
which is delayed 3 ms or more after the light is extinguished [4]. Since un-
couplers also inhibit the development of ion gradients across the lamellar mem-
branes, it was suspected that delayed fluorescence in some way depends on a
transmembrane hydrogen ion or salt concentration difference. This suspicion
was greatly strengthened by Mayne’s 1968 observation [5] that preilluminated
chloroplasts emit light when shifted from an acidic to a basic medium and by
Miles and Jagendorf’s 1969 observation [6] that abrupt increases in salt con-
centration cause a similar burst of fluorescence (see also ref. 7). Fleischmann
[8], Crofts et al. [9] and others suggested that photosynthetic electron tran-
sport establishes an electrochemical potential gradient across the membrane
and that this membrane potential lowers the activation energy required for the
recombination of the electron in Q‘ with a hole in an electron donor, Z‘. The
initial photochemical charge separations of photosynthesis probably occur
across the membrane, reducing Q to Q‘ toward the outside and oxidizing Z
to Z’ toward the inside [10]. If a membrane potential arises (positive to the
inside), it would then facilitate the return of an electron from Q' to Z’. To
explain the stimulation of delayed fluorescence by a pH gradient, Crofts et
al. [9] suggested that the electron-carrying redox couples Q/Q' and Z/Z+ are
in equilibrium with pools of secondary electron donors and acceptors which
are hydrogen-carrying redox couples. These pools are supposed to be them-
selves in equilibrium with the aqueous phases on the opposite sides of the
membrane and their redox potentials dependent on the pH of these phases.
Thus, the pH difference across the membrane could increase the availability
of either Q' or Z+ or both.

The first hint that the Fleischmann hypothesis might be wrong came from
the observations of Neumann et al. [11] and Felker et al. [12]. They showed
that, under certain conditions, uncoupling by ammonium salts enhanced
millisecond~delayed fluorescence instead of inhibiting it. However, it was by
no means certain that ammonia uncoupling actually abolished the membrane
potential. Furthermore, no conditions were found where other uncouplers
failed to inhibit millisecond-delayed fluorescence. Even methylamine, which
seems to uncouple in the same manner as ammonia, was always inhibitory.
Thus the Fleischmann model remained tenable.

We wish now to report an entirely different pattern of uncoupler effects on
delayed fluorescence which is observed when the measurements are made a few
microseconds after the excitation light is extinguished. The development of a
laser phosphoroscope which is capable of making reliable measurements of
delayed fluorescence within microseconds [13] allowed us to investigate the

96

4.48-

effects of uncouplers in this time range (see Methods).

It should be emphasized that our selection of a phosphoroscope was delibe-
rate and not motivated by the convenience of rapidly summing the signals.
We were particularly concerned about the relationship of delayed fluorescence
to the cumulative effects of frequently repeated periods of illumination on that
energized state of the membrane which is responsible for ATP formation and is
abolished by uncouplers. The concern arose from the fact that this state was
reputed to be involved in delayed fluorescence [8,9]. Single flash experiments
on dark-adapted chloroplasts would have provided us with no information on
these matters and, indeed, it is not at all clear why such single flash experi-
ments should be affected by uncouplers at all. It seemed to us that single flash
experiments on preilluminated chloroplasts would have suffered from them-
biguities of the phosphoroscope without the advantages. Both the phosphore-
scope and the single flash following preillumination have the disadvantage that
the delayed fluorescence following the period of illumination may be super-
imposed on the residual delayed fluorescence associated with the previous
period of illumination. On the other hand, the phosphoroscope has the added
advantage that we are able to measure electron transport and delayed fluores-
cence in the same experiment and this is patently impossible with a single flash.

The series of rapidly repeated illuminations with the strong light employed
in the laser phosphoroscope provided an average intensity capable of saturating
the electron transport system and, indeed, the rate of electron transport with
this intermittent light was no less than with continuous light of the same inten-
sity. Therefore, it is reasonable to suppose that the electron carrier pools at the
end of each millisecond illumination period were close to the conditions
achieved during steady-state electron transport.

We found that, in stimulating electron transport, uncouplers also increase the
intensity of the delayed fluorescence measured at any time between 5 and 100
us after light extinction. However, the rate of decline in the intensity of the
delayed fluorescence with time after light extinction is also increased by un-
couplers, so that within milliseconds uncouplers appear to inhibit delayed
fluorescence. These observations seem to preclude the possibility that any ener-
gized state of the membrane system which is sensitive to uncouplers can be
required for the delayed fluorescence reported here, be it a membrane poten-
tial, an ion gradient, or a transmembrane pH difference.
Materials and Methods a.

. ‘)‘w l‘

Chloroplast lamellae were isolated from commercial spinach by the method
of Ort and Izawa [14] and resuspended in a solution containing 0.2 M sucrose,
5 mM HEPES- NaOH (pH 7. 4) and 2 mM MgC12.

Nigericin and gramicidin were added in ethanol. The final ethanol concen-
tration in the reaction mixture was always less than 3%. This concentration of
ethanol had no effect on the phenomena being studied.

The laser phosphoroscope employed was a modification of that described)
by Beall and Haug [13]. Briefly, the light produced by a continuous output”
argon laser (principle lines at 488 and 515 nm) was focussed by a microscope]
objective and chopped by a rotating sector at the focal point. This gave a cycle».

97

449

of 1 ms light and 1 ms dark with a light-dark transition time of less than 250M

ms. The light beam was then collimated and directed through the bottom of a
1 X 1 X 4.5 cm quartz cuvette. Prompt and delayed fluorescence were measured
at a right angle to the actinic beam by a single photomultiplier tube cooled
with solid C02. A 690 nm interference filter was placed between the cuvette
and the photomultiplier to shield it from stray actinic light. The 50 SI output
of the photomultiplier was in discrete pulses 1--3 ns in length, each pulse asso-
ciated with individual photons striking the cathode surface. To measure the
delayed fluorescence, only those pulses generated during a specified period
after light extinction were counted. To do this its timing circuit was activated at
the instant of light extinction. This timing circuit created a counting “window”
by opening a gate from the photon detector to the counter for a prescribed
time. Under most circumstances this window began 5 us after light extinction
and ended 10 us after light extinction. The pulses arriving during 10 000 of
these gated counting periods were summed (one counting period every 2 ms for
20 s). The duration and time of commencement of the counting window could
be varied independently so that the rate of decay of delayed fluorescence could
be determined. When longer delays occurred before the fluorescence was mea-
sured, the rate of photon counting was so much slower that the window was
extended considerably, to 25 11s when the delay was 500 as and to 100 as when
the delay was 1 ms. However, in Figs. 1 and 2, the data presented have taken
into account this change in counting time. Prompt fluorescence was measured
with the same photomultiplier by allowing the gate to open to the counter
while the actinic light was still on.

Delayed fluorescence is reported as the ratio of counts during 5 us divided
by the prompt fluorescence in 5 us for the following reasons; we are interested
not so much in the actual delayed fluorescence as in the thermochemical reac-
tions responsible for the excitation of the chlorophyll. Since delayed fluores-
cence and prompt fluorescence seem to come from the same chlorophyll, it is
reasonable to assume that the chemically excited chlorophyll responsible for
delayed fluorescence fluoresces with the same efficiency as the light-excited

chlorophyll responsible for prompt fluorescence. Thus, dividing by the prompt

fluorescence should help to correct for variations in the fluorescence yield of
excited chlorophyll although, to be sure, the prompt fluorescence yield was not
measured and could not be measured at the same instant as the delayed fluores-
cence. Nevertheless, these “corrected” values for delayed fluorescence should
reflect the recombination reactions we wish to measure more accurately than
the delayed fluorescence itself. The “corrected” values have the added advan-
tage of normalizing the results of the various experiments since each data point
represents a separate experiment and there are inevitable small variations in the
actinic light intensity, chloroplast density and chloroplast condition.

In the same experiments electron transport was measured as the reduction of
ferricyanide. This was done by observing the changes in the ferricyanide absor-
bance at 420 nm. A weak light was passed through a 420 nm interference filter
before it passed through the cuvette. This beam was then detected with a
photomultiplier which was protected by another 420 nm interference filter to
screen out the actinic light. The signal from the photomultiplier was processed
by a logarithmic amplifier before it was fed into a strip chart recorder so that

 

98

450

rates of change in the concentration of ferricyanide could be directly deter-
mined. ' .5

In all cases the values given for delayed fluorescence intensity are the mean
of 5 determinations. Prompt fluorescence was measured after 20 s of preillu-
mination and immediately before and after the delayed fluorescence deter-
minations. All experiments were conducted at room temperature, approx.
23°C. The intensity of the light from the laser impinging on the reaction
cuvette was approximately 80 mW - cm". The reaction mixture occupied a
cubic volume 1 cm on each side.

The contrast ratio of the measurements of the apparent delayed fluorescence
and actual prompt fluorescence observed when there was really no delayed
fluorescence at all, was determined by replacing the chloroplast-containing
reaction mixture with a solution of chlorophyll in acetone. With this chloro-
phyll solution, the counts falsely attributed to the 5—10 us delayed fluores-
cence were always less than one per 10 000 counts of prompt fluorescence
measured over the same length of time. The intensity of the delayed 'fluores-
cence from chloroplasts was routinely ten times or more higher than this noise
level.

Results

Uncouplers of photophosphorylation and phosphorylating conditions
(ADP + P,) increase the intensity of delayed fluorescence for the first 100 as
after the light is turned off (Figs. 1 and 2). Ammonium ions routinely increase
the number of photons emitted 5—10 us after illumination about 3-fold.
Increases due to other uncouplers and phosphorylating conditions are some-
what smaller, viz., 50—100%. These stimulations of delayed fluorescence are
real and not artifacts of our method of correcting for the fluorescence effi-
ciency of excited chlorophyll (see Methods), since they are present and of a
similar magnitude whether or not such corrections are made. On the other
hand, the rate at which delayed fluorescence dies away after the light is off is
markedly increased by uncoupling and by ADP and Pi. Consequently un-
coupler-influenced delayed fluorescence is usually less than in the control after
about 150 us. As has been noted previously, delayed fluorescence disappears
almost entirely in 1 -2 ms with many uncouplers.

It is clear from these observations that the intensity of delayed fluorescence
cannot be dependent on any uncoupler-inhibited state of the membrane sys-
tem. On the contrary, some process which is enhanced by uncouplers or ADP
and Pi must be determining delayed fluorescence. Since all uncouplers share
with ADP + P, the ability to increase the electron transport [15] and the ability
to increase the intensity of 5-ps delayed fluorescence, we sought and found a
correlation between electron transport rates and the initial intensity of delayed
fluorescence.

Table I illustrates the correlation between electron transport and 5-ps
delayed fluorescence. In these experiments, electron transport was varied in
three ways: It was greatly diminished by omitting an exogenous electron
acceptor or almost abolished with the inhibitor DCMU. In both cases the inten-
sity of ps»delayed fluorescence was much lowered, in the case of DCMU almost

 

 

99

451

 

. 1
03‘i\ M40 <~\ NIGERICIN «
O

   

 

 

 

 

 

 

 

 

IL

0
1 -

8

_’ d>\“ 4
l. ‘\ GRAMICIDIN ‘
'1? ‘ d

\‘o‘“
‘-‘--m
l l_ l l l
ICC 500 IOOO ICC 500 IOOO

Time after light extinction (#5)

Fig. 1. Delayed fluorescence in spinach chloroplasts and its rate of decay in the presence and absence of
uncouplers. Reaction mixtures contained in 1.0 ml: chloroplasts with 5—10 pg chlorophyll; MgClz, 2
mo]: Tricine-NaOH (pH 7.8—8.1), 50 umol; potassium ferricyanide, 0.5 pmol; sucrose. 100 panel. When
added: NH401. 5 mol; methylamine-HG. 5 mol: gramicidin, 5 pg: nigericin, 5 pg. Ordinates are log
DF*, where DF" is 1000 times the ratio of the number of delayed fluorescence photons counted in 5
is in the dark to the number of prompt fluorescence photons counted in 5 us in the light. At 500 and
1000 us after the light was turned off, the periods of counting of delayed fluorescence photons were
increased to 25 and 100 1.8. respectively, with appropriate corrections. Solid lines represent decays of
delayed fluorescence in the presence of the uncoupler and dotted lines represent decays of delayed
fluorescence in the absence of the uncoupler. Note that the decay is polyphasic whether or not uncoup-
lers are present. This suggests that different reactions probably limit the back-reaction responsible for the
chemical excitation of chlorophyll at different times.

absent. Or electron transport was greatly increased by the use of uncouplers in
which case, as already noted, the increase in microsecond-delayed fluorescence
was marked. It is particularly important to note that the increase in delayed
fluorescence caused by uncouplers, which is noted in the presence of an exo-

 

 

 

 

06 I 1
«5
35
o. b q
\
s \
‘5 ‘\
o- \ ADP+ P, _
O
O
_J “
'03P -~‘~~().-- ‘- -4
......... '0
-06 1 1
ICC 500 I000

Time after light extinctionlps)
Fig. 2. Delayed fluorescence in spinach chloroplasts and its rate of decay in the presence and absence of

ADP and orthophosphate. Reaction conditions and data presentations as in Fig. 1. When added, ADP was
1.5 umol, and K2 HPO4. 5 umol.

.452

TABLE I

100

THE EFFECTS OF FERRICYANIDE, UNCOUPLERS, AND DCMU ON THE RELATIVE INTENSITIES
OF 5—10 MICROSECOND-DELAYED FLUORESCENCE AND ELECTRON TRANSPORT

Reaction conditions as in Fig. 1. DCMU when used was 100 nmol. Rates of electron transport are in umol
electrons - h‘l - mg"l Chl Rate of electron transport was measured with an oxygen electrode in a parallel
experiment as oxygen production in the presence of ferricyanide and as oxygen consumption in its

 

 

absence.
Additions Relative Electron
DF * transport

None 46 64
Methlyamine-HCl 46 6 5
Gramicidin 51 59
Ferricyanide 1 00 280
Ferricyanide, methylamine-HG! 187 1 170
Ferricyanide, gramicidin 1 47 1 240
Ferricyanide, DCMU 14 46

 

genous electron acceptor, does not occur when the electron transport rate is
limited by the absence of an exogenous electron acceptor. That is to say, un-
couplers do not increase the intensity of the us—delayed fluorescence when the
uncouplers fail to increase the electron transport rate.

Under some conditions, [us-delayed fluorescence is a nearly linear function
of the rate of electron transport (Fig. 3). However, this linear relationship may
only apply to a rather narrow range of special situations. For instance, the par-
tial inhibition of electron transport by DCMU, which blocks transport before

 

 

l
30-
3
o\ r 15’
g 20~ ,2" 'l
v ’1‘.
I
a
a
10- d
,DCMU

 

 

 

260 460 660 850
Electron Transport RaieUImole Ferricycnide reduced- hrrl- m9 Chl-'l
Fig. 3. The relationship of electron transport rate to the intensity of delayed fluorescence. In order to
lower the electron transport rate without lowering the light intensity. the chloroplasts were pretreated
with KCN, thus inactivating a portion of the plastocyanin [16]. A variety of electron transport rats were
achieved by varying the length of the KCN pretreatment (up to 48 min) and by uncoupling with Inefliyl-
amine. A, experiments with methylamine addition. Cyanide pretreatment was in an ice bath in the dark
with the following mixture: Tricine-NaOl-l (pH 8 .0). 100 mM; sucrose, 100 mM. MgClz. 1 mM; KCN. 50
mM; and potassium ferricyanide, 50 11M. Reaction mixture as in Fig.1. Methylamine-BC] when used‘. 5
mM. Numbers in parentheses represent minutes of incubation of the chloroplasts in the KCN. The valués
of the point labelled “DCMU” were calculated from the data of Table l.

 

101

453

 

5
o

u
0

OF *xLighi lniensulyt 5-IO/Isl

 

IZOO

600

 

Electron TronspoermoIes Ferricyamde reduced-hr"-mg Chl")

 

 

 

20 4O 60 80
Light Intensity (mw- cm’ 2)

Fig. 4. Delayed fluorescence and electron transport as functions of the exciting light intensity in the pres-
ence and absence of the uncoupler gramicidin. The value for “corrected” delayed fluorescence (DF*) is
obtained by dividing the observed delayed fluorescence by the prompt fluorescence. Since prompt
fluorescence under any one set of conditions is usually proportional to the incident light intensity, for the
purposes of the comparison made here it was necessary to multiply DI"I by the light intensity. The
maximum rate of electron transport in the absence of gramicidin was over 400 nmol ferricyanide reduce -
h"1 -mg‘1 Chl. which suggests that these chloroplasts had probably been inadvertantly partially un-
coupled.

plastoquinone, gives much more inhibition of ps-delayed fluorescence than
would be predicted from an extrapolation of the straight line in Fig. 3 where
the electron transport rate was varied by inhibiting plastocyanin. For that mat-
ter, this straight line does not extrapolate through the origin of the graph and,
therefore, it would be wrong to conclude that delayed fluorescence even
requires the net transfer of electrons to an exogenous acceptor. Furthermore,
different uncouplers sometimes give different intensities of delayed fluores-
cence at the same rate of reduction of the exogenous acceptor (data not
shown).

Changing the rate of electron transport by changing the light intensity has
very different effects on the delayed fluorescence (Fig. 4). Long after the light
intensity has been raised to a level where the electron transport system is satu-
rated, that is to say long after the rate of reduction of exogenous electron
acceptor has stopped increasing, the delayed fluorescence continuous to
increase. This is true whether or not the chloroplasts have been uncoupled by
gramicidin. However, regardless of the light intensity used, the uncoupler does
increase the 5-10-115 delayed fluorescence.

Discussion

The effects of uncouplers on microsecond-delayed fluorescence in chloro-
plasts have been observed by Jursinic et al. [17] using a single flash with and

 

 

 

102

454

without preillumination. These authors observed no inhibition with con—centra-
tions of gramicidin sufficient to abolish any membrane potential-induced absor-
bance change at 518 nm. They also observed that there was no enhancement
of 6—100 microsecond-delayed fluorescence in the presence of valinomycin in
response to an abrupt increase in KCI concentration. Consequently they'con-
cluded that membrane potentials probably did not play any part in providing
activation energy for microsecond-delayed fluorescence in their systems.

Our conclusions based on entirely different data are very similar. Uncouplers
which should diminish or abolish ion gradients and membrane potentials
actually increase microsecond-delayed fluorescence if electron transport is also
increased. It should be emphasized that the latter observations are not at
variance with the observations of Jurisinic et al. since their gramicidin effects
were observed in the absence of an electron acceptor and therefore in the
absence of a high rate of electron transport during preillumination. We also
found that gramicidin neither increased nor decreased microsecond-delayed
fluorescence in the absence of an exogenous electron acceptor (see Table I).

We also report that uncouplers and phosphorylating conditions increase the
rate of decay of the delayed fluorescence so that after milliseconds the delayed
fluorescence is diminished. Since the decay in the ability of chloroplasts to
produce delayed fluorescence is complex, it is difficult to determine the nature
of the processes involved in the decay. It seems likely that the delayed fluores-
cence measured microseconds after light extinction is the result of conditions
which are unrelated to the membrane potential but are intimately associated
with the rate of electron transport. It seems probable to us that the stimulation
of microsecond-delayed fluorescence and the increased rate of decay are both
due to some as yet unspecified effects of uncouplers of electron transport,
effects that determine the levels of the reactant species (Z, Q, P-680) arrived at
the light and also determine the rate at which they disappear in the dark by
non-radiative mechanisms.

Acknowledgements

This work was supported by National Science Foundation Grant 76-07581
to N .E. Good and by Energy Research and Development Administration Con-
tract No. EY—76-C-02-1338. The authors wish to thank B. Mayne and D.
Fleischmann for their comments on the manuscript and P. Jursinic for valuable
discussions.

References

Strehler, B.L. and Arnold, W. (1951) J. Gen. Physiol. 304, 809—820

Lavorel, J. (1975) in Bioenergetics of photosynthesis (Govindjee. ed.), pp. 223—317, Academic Press,
New York

Arthur, “(.15. and Strehler. B.L. (1957) Arch. Biochem. Biophys. 70, 507—526

Mayne, B. (1967) Photochem.,Photobiol. 6, 189—197

Mayne, B. (1968) Photochem. Photobiol. 8, 107—113

Miles, C.D. and Jagendorf. A. (1969) Arch. Biochem. Biophys. 129. 711

Barber. J. and Kraan, G.P.B. (1970) Biochim. Biophys. Acta 197, 49—95

Fleischmann, D.E. (1971) Photochem. Photobiol. 14, 277—286

Crofts. A.R.. Wraight, C.A. and Fleischmann, D.E. (1971) FEBS Lett. 15, 89—100

NH

@OQGOIfiW

 

10

11
12
13
14
15

16
17

103

455

Kraan. G.P.B., Amesz, J., Velthuys, B.R. and Steemers, R.G. (1970) Biochim. Biophys. Acta 223,
129-145

Neuman, J., Barber. J. and Gregory, P. (1973) Plant Physiol. 51, 1069—1073

Felker. P.. Izawa. 8.. Good, N.E. and Haug, A. (1974) Arch. Biochem. Biophys. 162, 345—356

Beall. H.C. and Haug, A. (1973) Anal. Biochem. 53, 98—107

Ort. D.R. and Izawa. S. (1973) Plant Physiol. 52, 595—600

Good, N.E. (1977) in Encyclopedia of Plant Physiology (Trebst, A. and Avron, M., eds.), Vol. 5.
pp. 429—447, Springer-Verlag, Berlin

Ort, D.R., Izawa, 8., Good, N.E. and Krogmann, D.W. (1973) FEBS Lett. 31, 119—122

Jursinic. P., Govindiee and Wraight, C.A. (1978) Photochem. Photobiol. 27, 61—71

APPENDIX II

ENERGY TRANSFER INHIBITION BY PYROPHOSPHATE

Appendix II

Pyr0phosphate, which has pK's of 5.8 and 8.2 is a
commonly-used buffer. In the course of some experiments
on ATP synthesis induced by an acid-base shift, one of the
students in our laboratory (T.Graan) thought to use it as
a buffer. Yields of ATP synthesis were abnormally low
under these circumstances, suggesting that the pyrophosphate
has some deleterious effects on phosphorylation.

I therefore attempted to establish whether this in-
hibition occurs with.steady-state phosphorylation. The
results presented in Figure l confirm that pyrophosphate
does indeed inhibit steady-state phosphorylation. A con-
centration of 2.5 mM was sufficient to inhibit phosphoryla-
tion 50% and coupled electron transport 38% while having no
effect on the basal electron transport or on the electron
transport uncoupled by gramicidin (not shown). Such effects
are typical of energy transfer inhibitors (such as phlorizin),
that is, agents which inhibit the mechanism of ATP synthesis
directly without any direct effects on electron transport.

In order to study more fully the nature of this in-
hibition, competition studies were carried out with Mgz+,
phosphate, and ADP. In contrast to phlorizin, which com-
petes with phosphate (Winget et al., 1969), pyrophosphate

seems to inhibit competitively with ADP (Figure 2).

104

105

Figure 1. Effect of pyrophoshate on the rates of phosphoryla-
ation and electron transport in spinach lamellae.
Reaction mixtures contained in 2.0 m1: chlorOplast
suspension containing 30 pg chlorophyll, 0.2 M
sorbitol, 2.0 mM MgC12, 10 mM Na2H 2po4, 10 mM KCl,

0.5 mM K3Fe(CN)6, 0.75 mM ADP, 50 mM Tricine-NaOH
(pH 8.0).

Solid lines indicates rates of electron transport
under non-phosphorylating (—P., filled circles)

and under phosphorylating conditions (open circles).
Dashed line indicates the effect of pyrophosphate
on the phosphorylation rate.

106

 

(complete)

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Concentration of Sodium Pyrophosphate

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(mM)

Figure 1.

107

 

   
   

(x1000)

1/ ATP rate

 

 

 

 

L l

 

0 0.5 1.0 2:0 3.3 5.0
1/ [ADP] (mM-l)

Figure 2. Double reciprocal plot of the inhibition
of phosphorylation by different levels
of pyrophoshate. Conditions as in
Figure l.

APPENDIX III
THE SUITABILITY OF SEVERAL NEW AMINOSULFONIC ACID

BUFFERS IN STUDIES OF PHOTOSYNTHESIS

Appendix III

In 1966, Good et a1. introduced a series of new buffers
that, because of their high water solubility and low
solubility in other solvents, low tendency to bind metals,
and pK's in a range important in biological reactions, have
become widely used. Several of these buffers were N-sub-
stituted aminoethane sulfonic acids and their wide-spread
use (especially of HEPES*) suggested the synthesis and use
of a series of N-substituted aminoeropane sulfonic acids
which were less expensive to synthesize (e.g. MOPS, TAPS,
HEPPS- Good and Izawa, 1972). However in recent years, it
has been determined that one of the precursors used in the
synthesis of these latter propane sulfonic buffers (propane
sultone) is carcinogenic and hazardous to use, and may soon
be commerically unavailable. This led Dr. William Ferguson,
a chemist at Research Organics Inc., Cleveland to prepare
a new series of buffers based on reactions with the

presumably harmless 3-chloro-2-propane-sulfonate. Five new

 

*HEPES N—hydroxyethylpiperazine-N'—ethanesulfonic acid

MOPS 3-(N-morpholino)propanesulfonic acid

MOPSO 3-(N-morpholino)—2-hydroxypropanesulfonic acid

HEPPS N—2-hydroxyethylpiperazine-N'-3—propanesulfonic acid

HEPPSO N-hydroxyethylpiperazine-N'-2-hydroxypr0pane-
sulfonic acid

TAPS 3-tris(hydroxymethyl)-methyl-3—aminopropanesulfonic
acid

TAPSO 3-N-tris(hydroxymethyl)methylamino-Z-hydroxy-
propanesulfonic acid

POPSO piperazine-N-N'-bis(2-hydroxypropanesulfonic acid)
dihydrate

DIPSO 3-N-bis(hydroxyethyl)amino—2-hydroxypropanesulfonic
acid 108

109

buffers were synthesized with pK's from 6.9 to 7.9. They
were MOPSO (pK 6.95), DIPSO (pK 7.6), TAPSO (pK 7.7)
HEPPSO (pK 7.9), and POPSO (pK 7.85).

This appendix reports that these new buffers are
satisfactory for phosphorylation and electron transport

studies in spinach lamellae. (Table I).

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APPENDIX IV
THE DESIGN OF AN INEXPENSIVE, RELIABLE, AND

RAPID-ACTING FRACTION COLLECTOR

Appendix IV

In the course of some experiments studying amine uptake
by the flow dialysis technique, a fast-acting fraction
collector was designed. 'Because of its ease of construction,
low cost, and reliability, this fraction collector may be of
general interest. The mechanical parts are illustrated in
Figure l.

The samples are collected in test tubes that are held
against the crown-shaped collector head by a band of rubber
cut from a rubber innertube. The V-shaped notches assure
very accurate positioning of the test tubes which may be of
any diameter equal to or less than the circumference of the
collector assembly divided by the number of notches. The
V-shaped notches do not extend the length of the collector
head but rather form a collar and a base which are separated
by removable spacers of a smaller diameter, thus allowing the
assembly to hold short or long test tubes of a wide variety
of diameters.

The drive mechanism for the collector is simple and
reliable. A 40 cm coiled door_spring is attached at one end
to a post and at the other end to a cord. This cord is

attached to the center shaft of the collector head. Turning

the head in a clockwise direction winds the cord on the shaft
and extends the spring. The taut spring then provides the

driving force turning the collector assembly. Positioning

111

112

Figure 1. Diagram of the escapement mechanism which
regulates the advancement of the fraction
collector.

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and advancement are performed by a simple escapement mech-
anism similar to that used in clocks and watches. Under
normal circumstances the return spring pulls on the end of
the pivoting escapement arm so that the forward end of the
arm is caught on a cog of the collector assembly. When it
is necessary to advance the fraction collector to the next
tube, a solenoid is actuated. This in turn pulls the
escapement arm and allows the collector assembly to advance
to a position where the motion is stopped by the second arm
of the escapement lever. This second arm of the escapement
lever prevents the collector assembly from advancing more than
one position while the solenoid is actuated. To complete the
advancement, the solenoid relaxes and the escapement arm
returns to the original position. The duration of a complete
advancement cycle takes only a few tenths of a second at most.
The design of the circuit that actuates the solenoid is
shown in Figure 2. Fractions can be collected for a preset
length of time or for a present number of drops. The drop
detector consists of a Light Emitting Diode (LED) and a
phototransitor with a gap between them through which a drOp
can pass. The action of the descending drop disperses the
light beam and momentarily lowers the voltage at the output
of the phototransistor. This signal is then amplified by a
transistor (02) and is fed into a comparator (one quarter of
an integrated circuit, 324). The integrated circuit compares
this voltage to a reference voltage. When the voltage from

Q2 is greater than the reference voltage the output of the

115

Figure 2. Schematic diagram of the fraction collector
circuit. All of the components were housed within
an aluminum box except the drop detector assembly
and the solenoid. For a discussion of the circuit,
see text.

Q1 —NPN phototransistor (Radio Shack 276-130)
Q , Q , Q -NPN transistor (MPS 2222A or equi—
2 4 5
valent)
Q3 —NPN power transistor(TIP 33)
Solenoid —type 322 (12 volt, 2.3 A)

LED Display -Seven-segment, common anode (Radio Shack
276-053)

116

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comparator is +5 volts, but when the voltage from Q2 is less
than the reference voltage, the output is zero. By proper
setting of the reference voltage, the action of a drOp
passing between the LED and the phototransistor is detected
at the output of the 325 as a brief excursion from +5 Volts
to zero and back to +5 volts. This signal is fed into a
one-shot multivibrator (pulse shaper) which lengthens its
duration and inverts it. The output of the pulse shaper is
fed into the counting circuit.

The heart of the counting circuit is a 74192 integrated
circuit. This is a presettable Up/Down decade counter which
can be set at any number from 0 to 9 as a binary—coded
decimal (BCD). A momentary grounding of pin 11 loads the
number that is in switches A,B,C, and D into the 74192. A?
pulse at pin 4 then causes the 74192 to count down to one
unit. If the previous number was zero, the 74192 goes from
zero to 9 and sends out a "borrow" pulse from pin 13. If
this borrow pulse is then fed into the count down input of the
next 74192, it is possible to count from 99 to zero. Adding
a third 74192 allows one to count from 999 to zero and so on.
The 7447 integrated circuit is a binary-coded decimal decoder
which decodes the BCD output of the 74192 for a common anode
7-segment LED display.

When zero is reached at all of the 74l92's the next
pulse would return them all to 9'3 and send out a pulse from

the "borrow" output of the third 74192 (most significant

118

digit). However if this pulse is fed into the "load" in-
puts of all of the 74192 integrated circuits, the counters
recycle to the present values rather than 999. This
borrow pulse also activates the solenoid-driving circuit
by triggering the one-shot multivibrator, a second 74121.
The setting of the pulse-width potentiometer determines the
duration of the solenoid actuation. The pulse from the
one-shot multivibrator makes transistors Q3, Q4, and Q5
conducting and discharges the 3300 HF capacitor through the
solenoid.

The power supply section of the circuit provides for
a +5 volt output (regulated by a heat-sinked integrated
circuit - 7805) and charges the 3300 uF capacitor to about
22 volts.

For time-based fraction collection, a one Hz pulse
is generated by a 555 integrated circuit. This pulse is
fed into the same pulse shaper as the drop-detecting circuit

and the counting proceeds as described above.

      

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